Compositions and methods for enhanced gene expression in cone cells

ABSTRACT

The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in cone cells.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling dates of U.S. Provisional Patent Application Ser. No. 61/954,330filed Mar. 17, 2014; and U.S. Provisional Patent Application Ser. No.62/127,185 filed Mar. 2, 2015, the full disclosures of which are hereinincorporated by reference.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is AVBI_(—)005_(—)02US_ST25.txt. The text file is308 KB, was created on Mar. 17, 2015, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

This invention pertains to gene therapy of retinal disorders.

BACKGROUND OF THE INVENTION

Vision disorders of the eye often relate to known primary defects incone cells. These include macular dystrophies such as Stargardt'smacular dystrophy, cone dystrophy, cone-rod dystrophy, Spinocerebellarataxia type 7, and Bardet-Biedl syndrome-1, as well as color visiondisorders, including achromotopsia, blue cone monochromacy, and protan,deutan, and tritan defects.

In addition to those disorders where the known cause is intrinsic tocone photoreceptors, there are vision disorders of the central macula(within primates) that may be treated by targeting cone cells. Theseinclude age-related macular degeneration, macular telangiectasia,retinitis pigmentosa, diabetic retinopathy, retinal vein occlusions,glaucoma, Sorsby's fundus dystrophy, adult vitelliform maculardystrophy, Best's disease, and X-linked retinoschisis.

A promising approach to treating and preventing ophthalmic disease thataddresses the limitations of existing treatment is delivery oftherapeutic agents to the eye with a gene therapy vector such as anadeno-associated virus (AAV). AAV is a 4.7 kb, single stranded DNAvirus. Recombinant vectors based on AAV are associated with excellentclinical safety, since wild-type AAV is nonpathogenic and has noetiologic association with any known diseases. In addition, AAV offersthe capability for highly efficient gene delivery and sustainedtransgene expression in numerous tissues, including eye, muscle, lung,and brain. Furthermore, AAV has shown promise in human clinical trials.One example is Leber's congenital amaurosis in which patients treatedwith a therapeutic delivered by a single subretinal administration of anrAAV vector have experienced sustained clinical benefit from expressionof the therapeutic agent for more than four years from the initial dateof treatment.

A number of challenges remain with regard to designing polynucleotidecassettes and expression vectors for use in gene therapy to treat eyedisease generally and cone cells specifically. One significant challengeis obtaining sufficient expression of the transgene in target cells,especially in cone cells of the retina. A longstanding unmet need in theart has been sufficiently robust expression of transgenes following genetransfer. In some cases, more efficient expression is required for theefficacy of certain vectors, for example plasmid DNA vectors. In othercases, more efficient gene expression cassettes are desirable to allowfor a lower therapeutic dose that has a more favorable safety profile ora less invasive route of administration (e.g., intravitreal vs.subretinal). In some settings, efficient expression has been achievedusing a strong, ubiquitous promoter, but it is often desirable to havehigh transgene expression using a nucleic acid expression cassette thatis only expressed in target cell types.

Previous efforts to express transgenes in cone cells, for example asdisclosed in US patent application US 2012/0172419, showed some promise,but often the expression levels were lower than optimal or not cellspecific. Given that a number of vision disorders result from primarydefects in cone cells, specific expression of transgenes in cone cells,with high expression levels, would represent a meaningful advance in theart. Therefore, there remains a need for improved methods and optimizednucleic acid cassettes and vectors for expressing genes in cone cells.

SUMMARY OF THE INVENTION

The present disclosure provides polynucleotide cassettes, expressionvectors and methods for the expression of a gene in cone cells.

In some aspects of the invention, polynucleotide cassettes are providedfor the expression of a transgene in cone cells of a mammalian retina.In some embodiments, the expression of the transgene is enhancedexpression. In certain embodiments, the expression of the codingsequence is greater than expression of the transgene operably linked toSEQ ID NO:1. In some embodiments, the expression of the transgene iscone-specific.

In some embodiments, the polynucleotide cassette comprises a promoterregion, wherein the promoter region promotes the expression of a gene inretinal cone cells; and a polyadenylation site. In some embodiments, theexpression is specifically in cone cells. In some such embodiments, thepromoter region comprises a polynucleotide sequence having a sequenceidentity of 85% or more to a sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:55, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81. SEQ ID NO:82,and SEQ ID NO:83, or a functional fragment thereof. In some embodiments,the promoter region is less than 492 nucleotides in length. In someembodiments, the promoter region consists essentially of apolynucleotide sequence having a sequence identity of 85% or more to thefull length of SEQ ID NO:55 or a functional fragment thereof.

In some embodiments, the polynucleotide cassette comprises apolynucleotide sequence encoding an untranslated region 5′ for a codingsequence, referred to herein as a 5′UTR. In some such embodiments, the5′UTR comprises a sequence having a sequence identity of 85% or more toa sequence selected from the group consisting of SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87, SEQ ID NO:88, and SEQ ID NO:89, or a fragment thereof. In someembodiments, some or all of the 5′UTR sequence is comprised by apromoter region as disclosed in, for example, SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:79.In some embodiments, the 5′UTR sequence is heterologous to the promotersequence. In some embodiments, the 5′UTR consists essentially of asequence having a sequence identity of 85% or more to the full length ofSEQ ID NO:85 or SEQ ID NO:86, or a fragment thereof. In someembodiments, the 5′UTR does not comprise a polynucleotide ATG.

In some embodiments, the polynucleotide cassette comprises an intron. Insome such embodiments, the intron comprises a sequence having a sequenceidentity of 85% or more to a sequence selected from the group consistingof SEQ ID NO:5, SEQ ID NO:59, and SEQ ID NO:60. In certain embodiments,the intron is located within the polynucleotide sequence encoding a5′UTR.

In some embodiments, the polynucleotide cassette comprises a translationinitiation sequence. In some such embodiments, the translationinitiation sequence comprises a polynucleotide sequence consistingessentially of SEQ ID NO:72 or SEQ ID NO:73.

In some embodiments, the polynucleotide cassette comprises an enhancersequence. In some such embodiments, the enhancer sequence comprises apolynucleotide sequence having a sequence identity of 85% or more to SEQID NO:52 or a functional fragment thereof. In certain embodiments, theenhancer sequence consists essentially of a sequence having a sequenceidentity of 85% or more to the full length of SEQ ID NO:51.

In some embodiments, the polynucleotide cassette comprises a codingsequence operably linked to the promoter. In some embodiments, thecoding sequence is heterologous to the promoter region and/or the 5′UTRsequence. In some embodiments, the coding sequence encodes a polypeptidehaving a sequence identity of at least 85%, 90%, or 95% to SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, and a polymorph of SEQ ID NO:11 selected from the groupconsisting of: (i) Thr65Ile (ii) Ile111Val (iii) Ser116Tyr (iv)Leu153Met (v) Ile171Val (vi) Ala174Val (vii) Ile178Val (viii) Ser180Ala(ix) Ile230Thr (x) Ala233Ser (xi) Val236Met (xii) Ile274Val (xiii)Phe275Leu (xiv) Tyr277Phe (xv) Val279Phe (xvi) Thr285Ala (xvii)Pro298Ala; and (xviii) Tyr309Phe. In some embodiments, the codingsequence has a sequence identity of at least 85%, 90%, or 95% to SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, or SEQ ID NO:71. In some embodiments, the sequencebetween the transcription initiation site and the end of coding sequencedoes not contain an open reading frame, other than the transgene openreading frame, that is more than 500 nucleotides in length. In someembodiments, the sequence between the transcription initiation site andthe end of coding sequence does not contain an open reading frame, otherthan the transgene open reading frame, that is more than 273 nucleotidesin length. In some embodiments, the sequence between the transcriptioninitiation site and the end of coding sequence does not contain an openreading frame, other than the transgene open reading frame, that is morethan 250 nucleotides in length. In some embodiments, at least one openreading frame of the coding sequence has been removed.

In some embodiments, the polynucleotide comprises a promoter region,wherein the promoter region promotes the expression of a gene in retinalcone cells; a 5′ untranslated region; an intron; a translationinitiation sequence; a coding sequence operatively linked to thepromoter region; and a polyadenlyation site. In some embodiments, thepolynucleotide comprises a promoter region, wherein the promoter regionpromotes the expression of a gene specifically in retinal cone cells; a5′ untranslated region; an intron; a translation initiation sequence; acoding sequence operatively linked to the promoter region; and apolyadenlyation site.

In some aspects of the invention, gene delivery vectors are providedcomprising a polynucleotide cassette of the present invention. In someembodiments, the gene delivery vector is a recombinant adeno-associatedvirus, wherein the recombinant adeno-associated virus comprises an AAVcapsid protein. In some embodiments, the AAV capsid protein is a wildtype AAV capsid protein. In other embodiments, the AAV capsid protein isa variant AAV capsid protein. In certain embodiments, the variant AAVcapsid protein comprises a peptide insertion in the AAV GH loop selectedfrom the group consisting of LGETTRP (SEQ ID NO:96), NETITRP (SEQ IDNO:97), KAGQANN (SEQ ID NO:98), KDPKTTN (SEQ ID NO:99), KDTDTTR (SEQ IDNO:100), RAGGSVG (SEQ ID NO:101), AVDTTKF (SEQ ID NO:102), and STGKVPN(SEQ ID NO:103).

In some aspects of the invention, pharmaceutical compositions areprovided comprising a polynucleotide cassette of the invention and apharmaceutical excipient. In some embodiments, the pharmaceuticalcomposition comprises a gene delivery vector of the invention and apharmaceutical excipient.

In some aspects of the invention, methods are provided for expressing atransgene in cone cells. In some embodiments, the method comprisescontacting one or more cone cells with an effective amount of apolynucleotide cassette of the invention or a gene delivery vector ofthe invention, wherein the transgene is expressed at detectable levelsin the one or more cone cells. In some embodiments, the method is invitro. In other embodiments, the method is in vivo. In certain suchembodiments, the contacting comprises injection of the polynucleotidecassette or gene delivery vector into the vitreous of a mammal eye. Inother such emobidments, the method comprises injection of thepolynucleotide cassette or gene delivery vector into the subretinalspace of a mammal eye. In some embodiments, the method further comprisesdetecting the expression of the trangene in cone cells, whereinexpression is detected in 80% or more of the cone cells. In someembodiments, the expression is specific for cone cells.

In some aspects of the invention, methods are provided for the treatmentor prophylaxis of a cone cell disorder in a mammal in need of treatmentor prophylaxis for a cone cell disorder. In some embodiments, the methodcomprises administering to the eye of the mammal an effective amount ofa pharmaceutical composition of the invention, wherein the codingsequence encodes a therapeutic gene product. In some embodiments, theadministering comprises injecting the pharmaceutical composition intothe vitreous of the mammal eye. In other such embodiments, the methodcomprises injecting the pharmaceutical composition into the subretinalspace of a mammal eye.

In some embodiments, the cone cell disorder is a color vision disorder.In certain embodiments, the color vision disorder is selected from thegroup consisting of achromotopsia, blue cone monochromacy, a protandefect, a deutan defect, and a tritan defect. In some such embodiments,the method further comprises detecting a change in the disease symptoms,wherein the change comprises an increase in the ability of the mammal toperceive a color. In some embodiments, the cone cell disorder is amacular dystrophy. In certain embodiments, the macular dystrophy isselected from the group consisting of Stargardt's macular dystrophy,cone dystrophy, cone-rod dystrophy, Spinocerebellar ataxia type 7, andBardet-Biedl syndrome-1. In some embodiments, the cone cell disorder isa vision disorder of the central macula. In certain embodiments, visiondisorder of the central macula is selected from the group consisting ofage-related macular degeneration, macular telangiectasia, retinitispigmentosa, diabetic retinopathy, retinal vein occlusions, glaucoma,Sorsby's fundus dystrophy, adult vitelliform macular dystrophy, Best'sdisease, rod-cone dystrophy, Leber's congenital amaurosis, and X-linkedretinoschisis. In some such embodiments, the method further comprisesdetecting a change in the disease symptoms. In some such embodiments,the change comprises a stabilization in the health of the cone cellsand/or a reduction in the rate of visual acuity loss of the mammal Incertain such embodiments, the change comprises an improvement in thehealth of the cone cells and/or an improvement in the visual acuity ofthe mammal

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A depicts the schematic overview of polynucleotide cassettes forenhanced expression of a transgene in cone cells.

FIG. 1B depicts a schematic overview of viral vectors comprisingpolynucleotide cassettes for enhanced expression of a transgene in conecells.

FIG. 2 depicts an example intron containing canonical features,including consensus sequences for the splice donor (A/C) A G G T Pu A GU; branch site C T Pu A Py; Py-rich region; and acceptor N C A G G.

FIG. 3A depicts an example 5′UTR mRNA structure (SEQ ID NO:56), the5′UTR mRNA structure from pR2.1 (Mancuso et al.). This 5′ UTR has twoupstream AUGs and open reading frames (ORF), a high level of basepairing and hairpin structure, and a shorter Kozak sequence.

FIG. 3B depicts an example 5′UTR mRNA and structure (SEQ ID NO:57) froman optimized cassette of the present disclosure. The 5′ UTR comprises noupstream AUG and no ORFs. In addition, as compared to the 5′ UTR of FIG.3A, the 5′ UTR of FIG. 3B is shorter, with less base pairing; and theKozak sequence is longer.

FIG. 4A depicts a polynucleotide cassette before codon optimization.Open reading frames (ORFs) greater than 250 nucleotides in length areshown in gray below the sequence.

FIG. 4B depicts a polynucleotide cassette after codon optimization, butbefore removal of non-transgene ORFs. ORFs greater than 250 nucleotidesare shown in gray below sequence diagram. Note the introduction of a newORF in reverse orientation beginning from SV40 polyA and extending 1,365bases.

FIG. 4C depicts a polynucleotide cassette after codon optimization andremoval of ORFs. ORFs greater than 250 nucleotides are shown in graybelow the sequence diagram. Note that the sequence has been optimized sothat newly introduced ORFs are shortened or removed.

FIG. 5 illustrates how intravitreally-delivered AAV2 variant AAV2-7m8transduces retinal cells in the fovea centralis and parafovea ofprimates more efficiently than intravitreally-delivered AAV2.5×10¹¹vector genomes of AAV2.CMV.GFP (upper left); AAV-2.5T.CMV.GFP (upperright) (Excoffon K. J., et al. 2009. Proc. Natl. Acad. Sci. U.S.A.106:3865-3870); (lower left) AAV2-7.8.CMV.GFP (Dalkara D, et al. SciTransl Med. 2013 Jun. 12; 5(189):189ra76); or AAV-ShH10.CMV.GFP (lowerright) (Klimczak R R et al. PLoS One. 2009 Oct. 14; 4(10):e7467) wasinjected into the vitreous of an African green monkey in a volume of 50uL, and GFP expression was observed 8 weeks later by OCT fluorescenceimaging in vivo.

FIG. 6 illustrates how robustly the pMNTC regulatory cassette promotesgene expression in foveal cones of primates. (a-b) AAV2-7m8.MNTC.GFP wasinjected into the central vitreous of a baboon and expression wasobserved (a) 5 weeks and (b) 8 weeks later by fundus fluorescence. (cand d) Natural GFP fluorescence within a 15 micron section of the foveaat approximately 6 months after injection with AAV2-7m8.MNTC.GFP at lowmagnification (c) and high magnification (d).

FIG. 7 illustrates robust and cone-specific gene expression in the conesof a mouse retina following intravitreal injection of AAV-deliveryMNTC.GFP. (a-b) Examples of GFP fluorescence 11 weeks after micereceived intravitreal injections of 5.04×10¹⁰ vector genomes viaintravitreal injection. (c-e) retinas were harvested for histology 14weeks after injection and cone outersegments were labeled with anantibody to L/M opsin (red). In (c) the red channel is turned off soonly the native GFP is visible, (d) is the same image with the redchannel on to allow visualization of cone outersegments. Comparison of(c) and (d) shows that most if not all cones were transduced by thevirus. (e) Image from the same retina as in c and d from different angleshowing profiles of cone photoreceptors.

FIG. 8 illustrates gene expression directed by the pMNTC regulatorycassette in the cones of the Mongolian gerbil retina. 1×10¹⁰-2×10¹⁰vector genomes of virus carrying GFP under the control of the CMV,pR2.1, or MNTC promoter were injected in a volume of 5 uL into thevitreous of a Mongolian gerbil, and GFP expression visualized at thedesignated time points by fundus fluorescence imaging. (a) Expression ofGFP directed by AAV2-7m8.CMV.GFP and AAV2-7m8.MNTC.GFP, visualized 4weeks after intravitreal administration. Gerbils 12-10, 12-11, and 12-12were injected with AAV2-7m8.CMV.GFP, while gerbils 12-13, 12-14, and12-15 were injected with AAV2-7m8.MNTC.GFP. OD, oculus dexter (righteye). OS, oculus sinister (left eye). (b) Expression of GFP directed byAAV2-7m8.pR2.1.GFP and AAV2-7m8.MNTC.GFP, 4 and 8 weeks later asdetected by fundus fluorescence imaging.

FIGS. 9A-9D demonstrate that the pMNTC regulatory cassette provides formore robust gene expression in foveal cones of primates than the conepromoter pR2.1. 5×10¹¹ vector genomes of AAV2-7m8.MNTC.GFP orAAV2-7m8.pR2.1.GFP were injected in a volume of 50 uL into the vitreousof African Green Monkeys as indicated (AAV2-7m8.MNTC.GFP into animals271 and 472; AAV2-7m8.pR2.1.GFP into animals 500 and 509). Retinas werevisualized in vivo at (a) 2 weeks, (b) 4 weeks, (c) 8 weeks, and (d) 12weeks for GFP using a fundus fluorescence camera (a, b, c, d) orautofluorescence on Heidelberg Spectralis OCT (a, b; data not shown forweeks 8 and 12). OD, oculus dexter (right eye). OS, oculus sinister(left eye).

FIGS. 10A-10D demonstrate the contribution of each of the optimizedpMNTC elements to the more robust expression observed. (a) The pMNTC andpR2.1 expression cassettes. (b) The experimental expression cassettes,in which each element in pMNTC is replaced one-by-one by thecorresponding element in pR2.1. (c,d) Expression of the luciferasetransgene in the retinas of gerbils intravitreally injected with each ofthe test articles (n=6-8 eyes per construct) as detected (c) 4 weeks and(d) 8 weeks after injection by IVIS imaging. “7m8.CMV” served as thepositive control.

DEFINITIONS

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Illustrative vectors include, for example, plasmids, viral vectors,liposomes, and other gene delivery vehicles.

The term “AAV” is an abbreviation for adeno-associated virus, and may beused to refer to the virus itself or derivatives thereof. The termcovers all subtypes and both naturally occurring and recombinant forms,except where required otherwise. The term “AAV” includes AAV type 1(AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAVtype 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8(AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV,non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infectprimates, “non-primate AAV” refers to AAV that infect non-primatemammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as a “rAAV vector particle” or simplya “rAAV vector”. Thus, production of rAAV particle necessarily includesproduction of rAAV vector, as such a vector is contained within a rAAVparticle.

The term “replication defective” as used herein relative to an AAV viralvector of the invention means the AAV vector cannot independentlyreplicate and package its genome. For example, when a cell of a subjectis infected with rAAV virions, the heterologous gene is expressed in theinfected cells, however, due to the fact that the infected cells lackAAV rep and cap genes and accessory function genes, the rAAV is not ableto replicate further.

An “AAV variant” or “AAV mutant” as used herein refers to a viralparticle composed of: a) a variant AAV capsid protein, where the variantAAV capsid protein comprises at least one amino acid difference (e g ,amino acid substitution, amino acid insertion, amino acid deletion)relative to a corresponding parental AAV capsid protein, and where thevariant capsid protein confers increased infectivity of a retinal cellcompared to the infectivity of the retinal cell by an AAV virioncomprising the corresponding parental AAV capsid protein, where the AAVcapsid protein does not comprise an amino acid sequence present in anaturally occurring AAV capsid protein; and b) a heterologous nucleicacid comprising a nucleotide sequence encoding a heterologous geneproduct.

The abbreviation “rAAV” refers to recombinant adeno-associated virus,also referred to as a recombinant AAV vector (or “rAAV vector”). A “rAAVvector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In general, the heterologous polynucleotide isflanked by at least one, and generally by two AAV inverted terminalrepeat sequences (ITRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids.

As used herein, the term “gene” or “coding sequence” refers to anucleotide sequence in vitro or in vivo that encodes a gene product. Insome instances, the gene consists or consists essentially of codingsequence, that is, sequence that encodes the gene product. In otherinstances, the gene comprises additional, non-coding, sequence. Forexample, the gene may or may not include regions preceding and followingthe coding region, e.g. 5′ untranslated (5′ UTR) or “leader” sequencesand 3′ UTR or “trailer” sequences, as well as intervening sequences(introns) between individual coding segments (exons).

As used herein, a “therapeutic gene” refers to a gene that, whenexpressed, confers a beneficial effect on the cell or tissue in which itis present, or on a mammal in which the gene is expressed. Examples ofbeneficial effects include amelioration of a sign or symptom of acondition or disease, prevention or inhibition of a condition ordisease, or conferral of a desired characteristic. Therapeutic genesinclude genes that correct a genetic deficiency in a cell or mammal

As used herein, a transgene is a gene that is delivered to a cell by avector.

As used herein, the term “gene product” refers to the desired expressionproduct of a polynucleotide sequence such as a polypeptide, peptide,protein or interfering RNA including short interfering RNA (siRNA),miRNA or small hairpin RNA (shRNA).

As used herein, the terms “polypeptide,” “peptide,” and “protein” referto polymers of amino acids of any length. The terms also encompass anamino acid polymer that has been modified; for example, disulfide bondformation, glycosylation, lipidation, phosphorylation, or conjugationwith a labeling component.

By “comprising” it is meant that the recited elements are required in,for example, the composition, method, kit, etc., but other elements maybe included to form the, for example, composition, method, kit etc.within the scope of the claim. For example, an expression cassette“comprising” a gene encoding a therapeutic polypeptide operably linkedto a promoter is an expression cassette that may include other elementsin addition to the gene and promoter, e.g. poly-adenylation sequence,enhancer elements, other genes, linker domains, etc.

By “consisting essentially of”, it is meant a limitation of the scope ofthe, for example, composition, method, kit, etc., described to thespecified materials or steps that do not materially affect the basic andnovel characteristic(s) of the, for example, composition, method, kit,etc. For example, an expression cassette “consisting essentially of agene encoding a therapeutic polypeptide operably linked to a promoterand a polyadenylation sequence may include additional sequences, e.g.linker sequences, so long as they do not materially affect thetranscription or translation of the gene. As another example, a variant,or mutant, polypeptide fragment “consisting essentially of” a recitedsequence has the amino acid sequence of the recited sequence plus orminus about 10 amino acid residues at the boundaries of the sequencebased upon the full length naïve polypeptide from which it was derived,e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the recitedbounding amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10residues more than the recited bounding amino acid residue.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim. For example, an expression cassette “consisting of” a geneencoding a therapeutic polypeptide operably linked to a promoter, and apolyadenylation sequence consists only of the promoter, polynucleotidesequence encoding the therapeutic polypeptide, and polyadenlyationsequence. As another example, a polypeptide “consisting of” a recitedsequence contains only the recited sequence.

As used herein, the terms “sequence identity,”e.g. “% sequenceidentity,” refers to the degree of identity between two or morepolynucleotides when aligned using a nucleotide sequence alignmentprogram; or between two or more polypeptide sequences when aligned usingan amino acid sequence alignment program. Similarly, the term“identical” or percent “identity” when used herein in the context of twoor more nucleotide or amino acid sequences refers to two sequences thatare the same or have a specified percentage of amino acid residues ornucleotides when compared and aligned for maximum correspondence, forexample as measured using a sequence comparison algorithm, e.g. theSmith-Waterman algorithm, etc., or by visual inspection. For example,the percent identity between two amino acid sequences may be determinedusing the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453)algorithm which has been incorporated into the GAP program in the GCGsoftware package, using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. As another example, the percent identity between twonucleotide sequences may be determined using the GAP program in the GCGsoftware package, using a NWSgapdna.CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5. The percent identity between two amino acid or nucleotidesequences can also be determined using the algorithm of E. Meyers and W.Miller (1989, Cabios, 4: 11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. The nucleic acid andprotein sequences described herein can be used as a “query sequence” toperform a search against public databases to, for example, identifyother family members or related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997, Nucleic Acids Res, 25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused.

The term “% homology” is used interchangeably herein with the term “%identity” herein and refers to the level of nucleic acid or amino acidsequence identity between two or more aligned sequences, when alignedusing a sequence alignment program. For example, as used herein, 80%homology means the same thing as 80% sequence identity determined by adefined algorithm, and accordingly a homologue of a given sequence hasgreater than 80% sequence identity over a length of the given sequence.

As used herein, the terms “complement” and “complementary” refer to twoantiparallel nucleotide sequences capable of pairing with one anotherupon formation of hydrogen bonds between the complementary base residuesin the antiparallel nucleotide sequences. For example, an shRNA might becomplementary, i.e. 100% complementary, or substantially complementary,e.g. 80% complementary, 85% complementary, 90% complementary, 95%complementary, 98% complementary, or more to a target sequence. The term“expression” as used herein encompasses the transcription and/ortranslation of an endogenous gene, a transgene or a coding sequence in acell.

An “expression vector” as used herein encompasses a vector, e.g.plasmid, minicircle, viral vector, liposome, and the like as discussedabove or as known in the art, comprising a polynucleotide which encodesa gene product of interest, and is used for effecting the expression ofa gene product in an intended target cell. An expression vector alsocomprises control elements operatively linked to the encoding region tofacilitate expression of the gene product in the target. The combinationof control elements, e.g. promoters, enhancers, UTRs, miRNA targetingsequences, etc., and a gene or genes to which they are operably linkedfor expression is sometimes referred to as an “expression cassette.”Many such control elements are known and available in the art or can bereadily constructed from components that are available in the art.

A “promoter” as used herein encompasses a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis, i.e., aminimal sequence sufficient to direct transcription. Promoters andcorresponding protein or polypeptide expression may be ubiquitous,meaning strongly active in a wide range of cells, tissues and species orcell-type specific, tissue-specific, or species specific. Promoters may“constitutive,” meaning continually active, or “inducible,” meaning thepromoter can be activated or deactivated by the presence or absence ofbiotic or abiotic factors. Also included in the nucleic acid constructsor vectors of the invention are enhancer sequences that may or may notbe contiguous with the promoter sequence. Enhancer sequences influencepromoter-dependent gene expression and may be located in the 5′ or 3′regions of the native gene.

An“enhancer” as used herein encompasses a cis-acting element thatstimulates or inhibits transcription of adjacent genes. An enhancer thatinhibits transcription also is termed a “silencer”. Enhancers canfunction (i.e., can be associated with a coding sequence) in eitherorientation, over distances of up to several kilobase pairs (kb) fromthe coding sequence and from a position downstream of a transcribedregion.

A “termination signal sequence” as used herein encompasses any geneticelement that causes RNA polymerase to terminate transcription, such asfor example a polyadenylation signal sequence.

A “polyadenylation signal sequence” as used herein encompasses arecognition region necessary for endonuclease cleavage of an RNAtranscript that is followed by the polyadenylation consensus sequenceAATAAA. A polyadenylation signal sequence provides a “polyA site”, i.e.a site on a RNA transcript to which adenine residues will be added bypost-transcriptional polyadenylation.

As used herein, the terms “operatively linked” or “operably linked”refers to a juxtaposition of genetic elements, e.g. promoter, enhancer,termination signal sequence, polyadenylation sequence, etc., wherein theelements are in a relationship permitting them to operate in theexpected manner. For instance, a promoter is operatively linked to acoding region if the promoter helps initiate transcription of the codingsequence. There may be intervening residues between the promoter andcoding region so long as this functional relationship is maintained. Asused herein, the term “heterologous” means derived from a genotypicallydistinct entity from that of the rest of the entity to which it is beingcompared. For example, a polynucleotide introduced by geneticengineering techniques into a plasmid or vector derived from a differentspecies is a heterologous polynucleotide. As another example, a promoterremoved from its native coding sequence and operatively linked to acoding sequence with which it is not naturally found linked is aheterologous promoter. Thus, for example, an rAAV that includes aheterologous nucleic acid encoding a heterologous gene product is anrAAV that includes a nucleic acid not normally included in anaturally-occurring, wild-type AAV, and the encoded heterologous geneproduct is a gene product not normally encoded by a naturally-occurring,wild-type AAV.

The term “endogenous” as used herein with reference to a nucleotidemolecule or gene product refers to a nucleic acid sequence, e.g. gene orgenetic element, or gene product, e.g. RNA, protein, that is naturallyoccurring in or associated with a host virus or cell.

The term “native” as used herein refers to a nucleotide sequence, e.g.gene, or gene product, e.g. RNA, protein, that is present in a wildtypevirus or cell. The term “variant” as used herein refers to a mutant of areference polynucleotide or polypeptide sequence, for example a nativepolynucleotide or polypeptide sequence, i.e. having less than 100%sequence identity with the reference polynucleotide or polypeptidesequence. Put another way, a variant comprises at least one amino aciddifference (e g , amino acid substitution, amino acid insertion, aminoacid deletion) relative to a reference polynucleotide sequence, e.g. anative polynucleotide or polypeptide sequence. For example, a variantmay be a polynucleotide having a sequence identity of 70% or more with afull length native polynucleotide sequence, e.g. an identity of 75% or80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99%identity with the full length native polynucleotide sequence. As anotherexample, a variant may be a polypeptide having a sequence identity of70% or more with a full length native polypeptide sequence, e.g. anidentity of 75% or 80% or more, such as 85%, 90%, or 95% or more, forexample, 98% or 99% identity with the full length native polypeptidesequence. Variants may also include variant fragments of a reference,e.g. native, sequence sharing a sequence identity of 70% or more with afragment of the reference, e.g. native, sequence, e.g. an identity of75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98%or 99% identity with the native sequence.

As used herein, the terms “biological activity” and “biologicallyactive” refer to the activity attributed to a particular biologicalelement in a cell. For example, the “biological activity” of an“immunoglobulin”, “antibody” or fragment or variant thereof refers tothe ability to bind an antigenic determinant and thereby facilitateimmunological function. As another example, the biological activity of apolypeptide or functional fragment or variant thereof refers to theabilty of the polypeptide or functional fragment or variant thereof tocarry out its native functions of, e.g., binding, enzymatic activity,etc. As a third example, the biological activity of a gene regulatoryelement, e.g. promoter, enhancer, kozak sequence, and the like, refersto the ability of the regulatory element or functional fragment orvariant thereof to regulate, i.e. promote, enhance, or activate thetranslation of, respectively, the expression of the gene to which it isoperably linked.

The terms “administering” or “introducing”, as used herein refer todelivery of a vector for recombinant protein expression to a cell, tocells and/or organs of a subject, or to a subject. Such administering orintroducing may take place in vivo, in vitro or ex vivo. A vector forexpression of a gene product may be introduced into a cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e. avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage).

“Transformation” is typically used to refer to bacteria comprisingheterologous DNA or cells which express an oncogene and have thereforebeen converted into a continuous growth mode such as tumor cells. Avector used to “transform” a cell may be a plasmid, virus or othervehicle.

Typically, a cell is referred to as “transduced”, “infected”;“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. The terms “transduced”, “transfected” and“transformed” may be used interchangeably herein regardless of themethod of introduction of heterologous DNA.

The term “host cell”, as used herein refers to a cell which has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof, e.g. reducing thelikelihood that the disease or symptom thereof occurs in the subject,and/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;or (c) relieving the disease, i.e., causing regression of the disease.The therapeutic agent may be administered before, during or after theonset of disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

The various compositions and methods of the invention are describedbelow. Although particular compositions and methods are exemplifiedherein, it is understood that any of a number of alternativecompositions and methods are applicable and suitable for use inpracticing the invention. It will also be understood that an evaluationof the expression constructs and methods of the invention may be carriedout using procedures standard in the art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology(including recombinant techniques), microbiology, biochemistry andimmunology, which are within the scope of those of skill in the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991), each ofwhich is expressly incorporated by reference herein.

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application.Nothing-herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further, the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ, conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides polynucleotide cassettes and expressionvectors for the expression of a gene in cone cells. Also provided aremethods for the use of these compositions in promoting the expression ofa gene in cone cells, for example, in an individual, e.g. for thetreatment or prophylaxis of a cone cell disorder. These and otherobjects, advantages, and features of the invention will become apparentto those persons skilled in the art upon reading the details of thecompositions and methods as more fully described below.

Compositions

In some aspects of the disclosure, compositions are provided for theexpression of a transgene in cone cells. By a “cone cell”, also referredto herein as a “cone photoreceptor” or “cone”, it is meant the subtypeof photoreceptor cells in the retina of the eye that function best inrelatively bright light. Cones are sensitive to specific wavelengths oflight and hence support the perception of color. In addition, conesrespond faster to stimuli than rod photoreceptors, perceiving finerdetail and more rapid changes in images than rods, and hence, supporthigh acuity vision for activities where visual detail is of primaryimportance such as reading and driving. Cones are readily identifiablein cross-sections of the retina by the cone-like shape of their outersegments. They are also readily identifiable by their location in theretina, the highest density of cones existing at the 1.5 mm depressionlocated in the center of the macula of the retina, called the “foveacentralis” or “foveal pit”.

In some embodiments of the disclosure, the composition that provides forthe expression of a transgene in cone cells is a polynucleotidecassette. By a “polynucleotide cassette” it is meant a polynucleotidesequence comprising two or more polynucleotide sequences, e.g.regulatory elements, translation initiation sequences, coding sequences,termination sequences, etc., typically in operably linkage to oneanother. Likewise, by a “polynucleotide cassette for the expression of atransgene in a cone cell,” it is meant a combination of two or morepolynucleotide sequences, e.g. promoter, enhancer, 5′UTR, translationinitiation sequence, coding sequence, termination sequences, etc. thatpromotes the expression of the transgene in a cone cell.

For example, in some embodiments, the polynucleotide cassette comprises:

-   -   (a) a promoter region, wherein the promoter region promotes the        expression of a coding sequence in cone cells; and    -   (b) a coding sequence operatively linked to the promoter region.        As another example, in some embodiments, the polynucleotide        cassette comprises:    -   (a) a promoter region, wherein the promoter region promotes the        expression of a coding sequence in retinal cone cells;    -   (b) a translation initiation sequence; and    -   (c) a coding sequence operatively linked to the promoter region.        As a third example, in some embodiments, the polynucleotide        cassette comprises:    -   (a) a promoter region, wherein the promoter region promotes the        expression of a coding sequence in retinal cone cells;    -   (b) a 5′ untranslated region;    -   (c) a translation initiation sequence; and    -   (d) a coding sequence operatively linked to the promoter region.        As a fourth example, in some embodiments, the polynucleotide        cassette comprises:    -   (a) a promoter region, wherein the promoter region promotes the        expression of a coding sequence in retinal cone cells;    -   (b) a 5′ untranslated region;    -   (c) an intron;    -   (d) a translation initiation sequence; and    -   (e) a coding sequence operatively linked to the promoter region.        As a fifth example, in some embodiments, the polynucleotide        cassette comprises:    -   (a) a promoter region, wherein the promoter region promotes the        expression of a coding sequence in retinal cone cells;    -   (b) a 5′ untranslated region;    -   (c) an intron;    -   (d) a translation initiation sequence; and    -   (e) a polyadenylation sequence.

In some embodiments, the polynucleotide cassettes of the presentdisclosure provide for enhanced expression of a transgene in cone cells.As demonstrated by the working examples of the present disclosure, thepresent inventors have discovered a number of polynucleotide elements,i.e. improved elements as compared to those known in the art, whichindividually and synergistically provide for the enhanced expression oftransgenes in cone cells. By “enhanced” it is meant that expression ofthe transgene is increased, augmented, or stronger, in cone cellscarrying the polynucleotide cassettes of the present disclosure relativeto in cone cells carrying the transgene operably linked to comparableregulatory elements, e.g. as known in the art. Put another way,expression of the transgene is increased, augmented, or stronger, fromthe polynucleotide cassettes of the present disclosure relative toexpression from a polynucleotide cassette not comprising the one or moreoptimized elements of the present disclosure, i.e. a reference control.For example, expression of the transgene is enhanced, or augmented, orstronger, in cone cells comprising a polynucleotide cassette comprisinga promoter disclosed herein than in cone cells that carry the transgeneoperably linked to a different promoter, e.g. as known in the art. Asanother example, expression of the transgene is enhanced, or increased,augmented, or stronger, in cone cells comprising a polynucleotidecassette comprising an enhancer sequence disclosed herein than in conecells that carry the transgene operably linked to a different enhancersequence. As another example, expression of the transgene is enhanced,or increased, augmented, or stronger, in cone cells comprising apolynucleotide cassette encoding a 5′UTR disclosed herein than in conecells that carry the transgene operably linked to a different 5′UTRcoding sequence. As another example, expression of the transgene isenhanced, or increased, augmented, or stronger, in cone cells comprisinga polynucleotide cassette comprising an intron as disclosed herein thanin cone cells that carry the transgene operably linked to a differentintronic sequence as known in the art. Exemplary sequences comprisingelements (e.g., promoters, enhancer sequences, 5′UTRs, and intons) thatmay be used as references for comparison include sequences encompassedby the native L-opsin promoter (SEQ ID NO:1) and variants thereof,sequences encompassed by the synthetic promoter pR2.1 (SEQ ID NO:50) andvariants thereof (e.g. pR1.7, pR1.5, pR1.1) as disclosed in, e.g. USApplication No. 2013/0317091, and sequences encompassed by theIRBP/GNAT2 promoter (US Applicaton No. 2014/0275231).

Without wishing to be bound by theory, enhanced expression of atransgene in cells is believed to be due to a faster build-up of geneproduct in the cells or a more stable gene product in the cells. Thus,enhanced expression of a transgene by the polynucleotide cassettes ofthe subject disclosure may be observed in a number of ways. For example,enhanced expression may be observed by detecting the expression of thetransgene following contact of the polynucleotide cassette to the conecells sooner, e.g. 7 days sooner, 2 weeks sooner, 3 weeks sooner, 4weeks sooner, 8 weeks sooner, 12 weeks sooner, or more, than expressionwould be detected if the transgene were operably linked to comparableregulatory elements, e.g. as known in the art. Enhanced expression mayalso be observed as an increase in the amount of gene product per cell.For example, there may be a 2-fold increase or more, e.g. a 3-foldincrease or more, a 4-fold increase or more, a 5-fold increase or more,or a 10-fold increase or more in the amount of gene product per conecell. Enhanced expression may also be observed as an increase in thenumber of cone cells that express detectable levels of the transgenecarried by the polynucleotide cassette. For example, there may be a2-fold increase or more, e.g. a 3-fold increase or more, a 4-foldincrease or more, a 5-fold increase or more, or a 10-fold increase ormore in the number of cone cells that express detectable levels of thetransgene. As another example, the polynucleotide of the presentinvention may promote detectable levels of the transgene in a greaterpercentage of cells as compared to a conventional polynucleotidecassette; for example, where a conventional cassette may promotedetectable levels of transgene expression in, for example, less than 5%of the cone cells in a certain region, the polynucleotide of the presentinvention promotes detectable levels of expression in 5% or more of thecone cells in that region; e.g. 10% or more, 15% or more, 20% or more,25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, insome instances 50% or more, 55% or more; 60% or more, 65% or more, 70%or more, or 75% or more, for example 80% or more, 85% or more, 90% ormore, or 95% or more of the cone cells that are contacted, will expressdetectable levels of gene product. Enhanced expression may also beobserved as an alteration in the viability and/or function of the conecells, e.g. as measured using assessment tools such as fundusphotography, OCT, adaptive optics, cERG, color vision tests, visualacuity tests, and the like, as known in the art and as described herein.

The polynucleotide cassettes of the present disclosure typicallycomprise a promoter region. Any suitable promoter region or promotersequence therein can be used in the subject polynucleotide cassettes, solong as the promoter region promotes expression of a coding sequence inretinal cone cells. In some embodiments, the promoter specificallypromotes expression of the gene in mammalian retinal cone cell; morepreferably primate retinal cone cells; more preferably in Catarrhiniretinal cone cells; even more preferably in human retinal cone cells. By“specifically” it is meant that the promoter predominately promotesexpression of the gene in the target cells as compared to other celltypes. Thus, for example, when a promoter region that specificallypromotes expression in cone cells is employed, more than 50% of theexpression, for example, at least any of 60%, 65%, 70% or 75% or more ofthe expression, e.g. at least any of 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 97%, 98%, 99%, 99.5%, or more of expression of the geneafter delivery of the subject polunucleotide cassette to the eye will bein cone cells.

Exemplary suitable promoter regions include the promoter region for anycone-specific gene, such as a 492 L-opsin promoter region (SEQ ID NO:1),a 491 L-opsin promoter region (SEQ ID NO:53), a 496 L-opsin promoterregion (SEQ ID NO:79), an M-opsin promoter region (SEQ ID NO:2, SEQ IDNO:54), a minimal M-opsin promoter region (SEQ ID NO:55, SEQ ID NO:93),a core M-opsin promoter sequence as disclosed for the first time herein(SEQ ID NO:80), an S-opsin promoter region (SEQ ID NO:3), an hRKpromoter region, and a cone arrestin promoter region; or portions orvariants thereof which retain activity promoting the expression of agene in cone cells. Nonlimiting examples of portions, or fragments, ofpromoter regions that find use in the subject polynucleotide cassetttesinclude promoter sequence immediately upstream of the 5′UTR, andcanonical binding sequences for transcription factors as known in theart. Such portions, or fragments, may be readily determined using anyconvenient method as known in the art or described herein. For example,the promoter sequence immediately upstream of the 5′UTR in SEQ ID NO:54and SEQ ID NO:55 may readily determined by in silico evaluation of thesequence as consisting essentially of nucleotides 1-406 of SEQ ID NO:54or nucleotides 1-154 of SEQ ID NO:55 using publicly available tools suchas, e.g. the UCSC genome BLAT browser; or by empirical testing throughoperable linkage with a reporter gene and introduction into cone cells,e.g. as described in the working examples herein. Shorter promotersequences are, in some embodiments, preferable to longer promotersequences, as they provide for more space in the vector for othernucleotide elements. In some embodiments, the promoter region is lessthan 492 base pairs in length. For example, in some embodiments, thefunctional fragment does not comprise nucleotides 1-10 or more of SEQ IDNO:1, for example, the functional fragment does not comprise nucleotides1-20 or more, nucleotides 1-30 or more, nucleotides 1-40 or more,nucleotides 1-50 or more of SEQ ID NO:1, e.g. nucleotides 1-60 or more,nucleotides 1-70 or more, nucleotides 1-80 or more, nucleotides 1-90 ormore, nucleotides 1-100 or more of SEQ ID NO:1, in some instancesnucleotides 1-120 or more, nucleotides 1-140 or more, nucleotides 1-160or more, nucleotides 1-180 or more, nucleotides 1-200 or more,nucleotides 1-220 or more, nucleotides 1-240 or more, or aboutnucleotides 1-260 of SEQ ID NO:1. Any suitable method for identifying apromoter region capable of driving expression in mammalian or primatecone cells can be used to identify promoter regions and promotersequences therein that find use in the polynucleotide cassettes of thepresent disclosure.

In some embodiments, the promoter region of the subject polynucleotidecassette comprises one of the promoter regions disclosed herein, e.g. a492 L-opsin promoter region (SEQ ID NO:1), a 491 L-opsin promoter region(SEQ ID NO:53), a 496 L-opsin promoter region (SEQ ID NO:79), an M opsinpromoter region (SEQ ID NO:2, SEQ ID NO:54), a minimal M opsin promoterregion (SEQ ID NO:55, SEQ ID NO:93), the core M-opsin promoter sequencedisclosed herein (SEQ ID NO:80), or the S opsin promoter region (SEQ IDNO:3), or a functional fragment or variant thereof, e.g. a sequencehaving an identity of 75% or more, e.g. 80% or more, 85% or more, 90% ormore, or 95% or more, (e.g., 80%, 85%, 90% Or 95%), to an aforementionedsequence or functional fragment thereof. In some embodiments, thepromoter sequence of the subject polynucleotide cassette consistsessentially of one of the promoter regions disclosed herein, i.e. SEQ IDNO:1, SEQ ID NO:53, SEQ ID NO:79, SEQ ID NO:2, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:93, SEQ ID NO:80, or SEQ ID NO:3, or a functionalfragment or variant thereof, e.g. a sequence having an identity of 75%or more, e.g. 80%, or more 85% or more, 90% or more, or 95% or more,(e.g., 80%, 85%, 90% Or 95%), to the full length of an aforementionedsequence plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, orfunctional fragment thereof. In some embodiments, the promoter region ofthe subject polynucleotide cassette consists of one of the promoterregions disclosed herein, i.e. SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:79,SEQ ID NO:2, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:93, SEQ ID NO:80, orSEQ ID NO:3, or a functional fragment or variant thereof, e.g. asequence having an identity of 75% or more, e.g. 80%, 85%, 90%, 95% ormore, to the full length of an aforementioned sequence or functionalfragment thereof. In certain embodiments, the promoter region consistsessentially of SEQ ID NO:74. In some such embodiments, the promotersequence consists essentially of SEQ ID NO:80. In some embodiments, thepromoter results in enhanced expression in cone cells compared to otherpromoters known in the art, e.g., the synthetic promoters pR2.1,pR1.7,pR1.1, and IRBP/GNAT2.

In some embodiments, the polynucleotide cassette further comprises anenhancer element. Enhancers are nucleic acid elements known in the artto enhance transcription, and can be located anywhere in associationwith the gene they regulate, e.g. upstream, downstream, within anintron, etc. Any enhancer element can be used in the polynucleotidecassettes and gene therapy vectors of the present disclosure, so long asit enhances expression of the gene when used in combination with thepromoter. In a preferred embodiment, the enhancer element is specificfor retinal cone cells; more preferably, it is specific for primateretinal cone cells; more preferably in Catarrhini retinal cone cells;even more preferably in human retinal cone cellsBy “specifically” it ismeant that the enhancer predominately enhances expression of the gene inthe target cells compared to other cell types. Thus, for example, whenan enhancer that specifically enhances expression in cone cells isemployed, more than 50% of the expression, for example, at least any of60%, 65%, 70%, 75% or more of the expression, e.g., at least 80%, andpreferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99%,99.5%, or more of expression of the gene after delivery of the vector tothe eye will be in cone cells.

Exemplary enhancer regions that find use in the polynucleotide cassettesof the present disclosure include those that comprise, consistessentially of, or consist of the enhancer region for any cone-specificgene or fragments or variants thereof which retain enhancer activity.For example, the L/M minimal opsin enhancer, referred to as the LocusControl Region (LCR) (Wang et al., 1992. Neuron 9: 429-440) (SEQ IDNO:52) can be used to enhance gene expression in cone cells; its absenceresults in blue cone monochromacy (Nathans et al., 1989; Science, 245:831-838). The LCR has been shown to be useful in gene therapy, forexample with AAV vectors (Li et al., Vision Research 48(2008): 332-338).Furthermore, a functional fragment consisting essentially of a 36 by“core” LCR sequence has been identified that is necessary and sufficientfor expression from the opsin promoter in cone cells (Komaromy et al.Targeting gene expression to cones with human cone opsin promoters inrecombinant AAV. Gene Ther. 2008; 15(14):1049-55) (SEQ ID NO:51). Insome embodiments, the enhancer of the polynucleotide cassette comprisesSEQ ID NO:51 or SEQ ID NO:52. In certain embodiments, the enhancer ofthe polynucleotide cassette consists essentially of SEQ ID NO:51 or SEQID NO:52.

L/M enhancer elements of 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, ormore nucleotides that comprise one or more copies of the L/M minimalopsin enhancer, and the full L/M opsin enhancer, or other portions orvariants thereof which retain activity enhancing expression of genes ina cone-specific manner find use in the present compositions. Anysuitable method for identifying enhancer sequences capable of drivingexpression in primate cone cells can be used to identify such enhancers,as will be understood by those of skill in the art based on theteachings herein.

The length of the promoter and enhancer regions can be of any suitablelength for their intended purpose, and the spacing between the promoterand enhancer regions can be any suitable spacing to promotecone-specific expression of the gene product. In various preferredembodiments, the enhancer is located 0-1500; 0-1250; 0-1000; 0-750;0-600; 0-500; 0-400; 0-300; 0-200; 0-100; 0-90; 0-80; 0-70; 0-60; 0-50;0-40; 0-30; 0-20; or 0-10 nucleotides upstream of the promoter. Thepromoter can be any suitable distance upstream of the encoded gene.

In some embodiments, the subject polynucleotide cassette comprises asequence encoding a 5′ untranslated region, i.e. polynucleotide sequenceencoding an untranslated region 5′ to the coding sequence, also calledthe 5′UTR. In an expression cassette, the 5′UTR is known in the art asthe sequence between the transcription initiation site and the Kozaksequence where protein translation begins. Secondary mRNA structure ofthe 5′UTR is known to affect transcription levels. Specifically, forenhanced gene expression, the sequence of the 5′UTR region in thepresent invention is selected to minimize or avoid secondary structuresand upstream AUG (uAUG) codons which are known to decrease translationefficiency due to inefficient ribosome scanning and false translationalstarts (Kozak, 1995. PNAS 92:2662). See Davuluri et al., GenomeResearch, 2000: 10 (11); 1807-1816. For example, the 5′UTR sequence fromthe human gene HSP70 (SEQ ID NO:58) has been identified for its unusualability to enhance mRNA translation, possibly due to an IRES mechanism(Rubtsova et al., 2003. PNAS 278(25): 22350-22356; Vivinus et al, 2001.Eur J Biochem. 268: 1908-1917). Any 5′ UTR can be used, but ideally thesequence of the 5′UTR has minimal secondary mRNA structure and upstreamAUG sequences. Put another way, in some embodiments, the sequencebetween the transcription initiation site and the translation initiationsite of the polynucleotide cassette does not contain the polynucleotideATG. In some embodiments, the 5′ UTR comprises, consists essentially, orconsists of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:84, SEQID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, or SEQ ID NO:89; ora functional fragment or variant thereof, for example, a polynucleotidesequence having a sequence identity of 85% or more to a sequenceselected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ IDNO:88, and SEQ ID NO:89, or a fragment thereof. In some embodiments,some or all of the 5′UTR sequence is comprised by a promoter region asdisclosed in, for example, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:79. In otherembodiments, the 5′UTR is not comprised by the promoter region; see,e.g. the core promoter sequence SEQ ID NO:84, which does not encode for5′ UTR sequence. In some embodiments, the 5′UTR sequence is heterologousto the promoter sequence. In various preferred embodiments, the 3′ endof the UTR is 0-20; 0-15; 0-10; 0-9; 0-8; 0-7; 0-6; or 0-5 nucleotidesupstream of the coding sequence, and its 5′ end is 0-20; 0-15; 0-10;0-9; 0-8; 0-7; 0-6; or 0-5 nucleotides downstream of the proximalpromoter region. In some embodiments, the 5′UTR element results inenhanced expression in cone cells compared to other 5′UTRs known in theart, e.g., the 5′UTRs comprised by the synthetic promoters pR2.1, pR1.7,pR1.1, and IRBP/GNAT2.

In some embodiments, the subject polynucleotide cassette furthercomprises an intron comprising a splice donor/acceptor region. In someembodiments, the intron is located downstream of the promoter region andis located upstream of the translation initiation sequence of the gene,i.e. the intron is located within the 5′UTR. In other embodiments, theintron is located downstream of the translation initiation sequence ofthe gene, i.e. the intron is located within the coding sequence. As isgenerally known in the art, introns are DNA polynucleotides that aretranscribed into RNA and removed during mRNA processing through intronsplicing. Polynucleotide cassettes containing introns generally havehigher expression than those without introns. Introns can stimulateexpression between 2- and 500-fold (Buchman and Berg, 1988. Mol Cel Bio,8(10): 4395). Efficiently spliced introns contain a pre-splice donor,branchpoint, and Py rich region (Senapathy et al, 1990; Meth. Enzymol.183, 252-78; Wu and Krainer, 1999; Mol Cell Biol 19(5):3225-36). 5′introns are generally more efficient compared to introns at the 3′ end(Huang and Gorman, 1990; Mol Cell Bio, 10:1805). Although introns areknown generally to increase the level of gene expression, the specificincrease (if any) of a given cDNA is empirical and must be tested; forexample the chimeric intron in the pSI vector increases CAT expressionby 21-fold, but luciferase expression by only 3-fold.

Any intron can be used in the subject polynucleotide cassettes, so longas it comprises a splice donor/acceptor region recognized in mammalianor in primate cone cells, so that the intron can be spliced out of theresulting mRNA product. In one embodiment, the intron comprises,consists essentially of, or consists of an SV40 intron according to SEQID NO:5. In another embodiment, the intron comprises, consistsessentially of, or consists of the chimeric intron from pSI (SEQ IDNO:60) or a variant thereof. In another embodiment, the introncomprises, consists essentially of, or consists of the CMV intron A or avariant thereof. In yet another embodiment, the intron comprises,consists essentially of, or consists of the pR2.1 intron (SEQ ID NO:59)or a variant thereof, or alternatively, the rabbit or human beta globinintron (Xu et al, 2001, Gene 272:149; Xu et al.2002; J Control Rel81:155) or a variant thereof. In some such embodiments, the introncomprises a sequence having a sequence identity of 85% or more to asequence selected from the group consisting of SEQ ID NO:5, SEQ IDNO:59, and SEQ ID NO:60. Typically, the intron is heterologous to thepromoter region and/or the 5′UTR.

In some embodiments, the intron resides within a 5′UTR. In other words,the DNA sequence encoding the 5′UTR is interrupted by intronic DNAsequence. For example, the coding sequence for the 5′UTR that is SEQ IDNO:84 may be encoded in two parts, e.g. SEQ ID NO:85 and SEQ ID NO:86,with an intronic sequence between them. As another example, the codingsequences for the 5′UTR that is SEQ ID NO:88 may be encoded in twoparts, e.g. SEQ ID NO:89 and SEQ ID NO:73, with an intronic sequencebetween them. In various embodiments, the 3′ end of the intron is 0-20;0-15; 0-10; 0-9; 0-8; 0-7; 0-6; or 0-5 nucleotides upstream of the gene,and its 5′ end is 0-20; 0-15; 0-10; 0-9; 0-8; 0-7; 0-6; or 0-5nucleotides downstream of the proximal promoter region. In otherembodiments, the intron resides within the coding sequene of the gene.

In some embodiments, the polynucleotides cassettes of the presentdisclosure comprise a translation initiation sequence, also know as a“Kozak sequence” or “Kozak translation initiation sequence. This is thenucleic acid sequence where the ribosome attaches and translationbegins. Examples include ACCATGG (Kozak, 1986. Cell, 44:283-292) and(GCC)GCC(A/G)CCATGG (Kozak, 1987. Nucl Acid Res; 15(20): 8125) (SEQ IDNO:73). Any suitable Kozak sequence can be used in the polynucleotidecassette, preferably selected to increase expression of the codingsequence in retinal cone cells. In one embodiment, the translationinitiation sequence comprises SEQ ID NO:72. In an alternativeembodiment, the translation initiation sequence comprises SEQ ID NO:73.In some embodiments, the Kozak element results in enhanced expression incone cells compared to other Kozak sequences known in the art, e.g., theKozak sequences comprised by the synthetic promoters pR2.1, pR1.7,pRl.1, and IRBP/GNAT2.

In some aspects of the present invention, the subject polynucleotidecassettes are used to deliver a gene to cone cells of an animal, e.g. todetermine the effect that the gene has on cell viability and/orfunction, to treat a cone cell disorder, etc. Accordingly, in someembodiments, the polynucleotide cassettes of the present disclosurefurther comprise a gene to be delivered as a transgene to cone cells ofan animal in vitro or in vivo. The gene coding sequence is typicallyoperatively linked to the promoter region of the subject polynucleotidecassette, and in instances in which an an enhancer element is present,to the enhancer element of the subject polynucleotide cassette, suchthat the promoter and optionally enhancer elements promote theexpression of the coding sequence or cDNA in cone cells of the subject.

The coding sequence to be expressed in the cone cells can be anypolynucleotide sequence, e.g. gene or cDNA that encodes a gene product,e.g. polypeptide or RNA-based therapeutic (siRNA, antisense, ribozyme,shRNA, etc.). The coding sequence may be heterologous to the promotersequence and/or 5′UTR sequence to which it is operably linked, i.e. notnaturally operably associated with it. Alternatively, the codingsequence may be endogenous to the promoter sequence and/or 5′UTRsequence to which it is operably linked, i.e. is associated in naturewith that promoter or 5′UTR. The gene product may act intrinsically inthe cone cell, or it may act extrinsically, e.g. it may be secreted. Forexample, when the transgene is a therapeutic gene, the coding sequenemay be any gene that encodes a desired gene product or functionalfragment or variant thereof that can be used as a therapeutic fortreating a cone cell disease or disorder, or as a means to otherwiseenhance vision, including but not limited to promoting tetrachromaticcolor vision. In various preferred embodiments, the transgene encodes atherapeutic protein or functional fragment or variant thereof selectedfrom the group consisting of:

-   -   (a) SEQ ID NO:7 (SEQ ID NO:6) Homo sapiens opsin 1 (cone        pigments), short-wave-sensitive (OPN1SW), mRNA NCBI Reference        Sequence: NM_(—)001708.2;    -   (b) SEQ ID NO:9 (SEQ ID NO:8) Homo sapiens opsin 1 (cone        pigments), medium-wave-sensitive (OPN1MW), mRNA NCBI Reference        Sequence: NM_(—)000513.2;    -   (c) SEQ ID NO:11 (SEQ ID NO:10) Homo sapiens opsin 1 (cone        pigments), long-wave-sensitive (OPN1LW), mRNA NCBI Reference        Sequence: NM_(—)020061.4;    -   (d) SEQ ID NO:13 (SEQ ID NO:12) ATP binding cassette retina gene        (ABCR) gene (NM_(—)000350);    -   (e) SEQ ID NO:15 (SEQ ID NO:14) retinal pigmented        epithelium-specific 65 kD protein gene (RPE65) (NM._(—)000329);    -   (f) SEQ ID NO:17 (SEQ ID NO:16) retinal binding protein 1 gene        (RLBP1) (NM._(—)000326);    -   (g) SEQ ID NO:19 (SEQ ID NO:18) peripherin/retinal degeneration        slow gene, (NM_(—)000322);    -   (h) SEQ ID NO:21 (SEQ ID NO:20) arrestin (SAG) (NM_(—)000541);    -   (i) SEQ ID NO:23 (SEQ ID NO:22) alpha-transducin (GNAT1)        (NM_(—)000172);    -   (j) SEQ ID NO:24 guanylate cyclase activator 1A (GUCA1A)        (NP_(—)000400.2);    -   (k) SEQ ID NO:25 retina specific guanylate cyclase (GUCY2D),        (NP_(—)000171.1);    -   (l) SEQ ID NO:26 & 27 alpha subunit of the cone cyclic        nucleotide gated cation channel (CNGA3) (NP_(—)001073347.1 or        NP_(—)001289.1);    -   (m) SEQ ID NO:28 Human cone transducin alpha subunit (incomplete        achromotopsia);    -   (n) SEQ ID NO:29 cone cGMP-specific 3′,5′-cyclic        phosphodiesterase subunit alpha′, protein (cone dystrophy type        4);    -   (o) SEQ ID NO:30 retinal cone rhodopsin-sensitive cGMP        3′,5′-cyclic phosphodiesterase subunit gamma, protein (retinal        cone dystrophy type 3A);    -   (p) SEQ ID NO:31 cone rod homeobox, protein (Cone-rod        dystrophy);    -   (q) SEQ ID NO:32 cone photoreceptor cyclic nucleotide-gated        channel beta subunit, protein (achromatopsia);    -   (r) SEQ ID NO:33 cone photoreceptor cGMP-gated cation channel        beta-subunit, protein (total color blindness, for example, among        Pingelapese Islanders);    -   (s) SEQ ID NO:35 (SEQ ID NO:34) retinitis pigmentosa 1        (autosomal dominant) (RP 1);    -   (t) SEQ ID NO:37 (SEQ ID NO:36) retinitis pigmentosa GTPase        regulator interacting protein 1 (RPGRIP 1);    -   (u) SEQ ID NO:39 (SEQ ID NO:38) PRP8;    -   (v) SEQ ID NO:41 (SEQ ID NO:40) centrosomal protein 290 kDa        (CEP290);    -   (w) SEQ ID NO:43 (SEQ ID NO:42) IMP (inosine 5′-monophosphate)        dehydrogenase 1 (IMPDH1), transcript variant 1;    -   (x) SEQ ID NO:45 (SEQ ID NO:44) aryl hydrocarbon receptor        interacting protein-like 1 (AIPL1), transcript variant 1;    -   (y) SEQ ID NO:47 (SEQ ID NO:46) retinol dehydrogenase 12        (all-trans/9-cis/11-cis) (RDH12);    -   (z) SEQ ID NO:49 (SEQ ID NO:48) Leber congenital amaurosis 5        (LCAS), transcript variant 1; and    -   (aa) exemplary OPN1LW/OPN1MW2 polymorphs (compared to OPN1LW (L        opsin) polypeptide sequence; the amino acid to the left of the        number is the residue present in the L opsin sequence; the        number is the reside number in L opsin, and the reside to the        right of the number is the variation from L opsin. Polymorphs        according to these embodiments may comprise one or more of the        amino acid substitutions selected from Thr65Ile; Ile111Val;        Ser116Tyr; Leu153Met; Ile171Val; Ala174Val; Ile178Val;        Ser180Ala; Ile230Thr; Ala233Ser; Val236Met; Ile274Val;        Phe275Leu; Tyr277Phe; Val279Phe; Thr285Ala; Pro298Ala;        Tyr309Phe;    -   (ab) Additional Opsin Sequence Variation 1 (SEQ ID NO:61);    -   (ac) Additional Opsin Sequence Variation 2 (SEQ ID NO:62);    -   (ad) Additional Opsin Sequence Variation 3 (SEQ ID NO:63);    -   (ae) Additional Opsin Sequence Variation 4 (SEQ ID NO:64);    -   (af) Additional Opsin Sequence Variation 5 (SEQ ID NO:65);    -   (ag) Additional Opsin Sequence Variation 6 (SEQ ID NO:65);    -   (ah) Additional Opsin Sequence Variation 7 (SEQ ID NO:66);    -   (ai) Additional Opsin Sequence Variation 8 (SEQ ID NO:67);    -   (aj) Additional Opsin Sequence Variation 9 (SEQ ID NO:68);    -   (ak) hCHR2 (channel rhodopsin) (SEQ ID NO:69);    -   (al) NpHR (halorhodopsin) (SEQ ID NO:70); and    -   (am) eGFP (SEQ ID NO:71).

In some embodiments, the coding sequence encoded by the transgeneencodes a polypeptide having at least 85% sequence identity to apolypeptide encoded by a sequence disclosed above or herein, for exampleat least 90% sequence identity, e.g. at least 95% sequence identity, atleast 98% sequence identity, or at least 99% sequence identity. Thus,for example, the coding sequence encodes a cone opsin having at least85%, at least 90%, at least 95% identity, at least 98% sequenceidentity, or at least 99% sequence identity, to the polypeptide encodedby OPN1LW, OPNIMW, or OPN1SW. In some embodiments, the coding sequencehas a sequence identity of at least 85%, 90%, 95%, 98% or at least 99%to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34,SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44,SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.

The proteins recited in (a)-(c) and (aa-aj) are all involved in colorvision. The exemplary polymorphs include ones at positions 65, 116, 180,230, 233, 277, 285, and 309 that affect the spectra of the pigments incone cells expressing them. Positions 274, 275, 277, 279, 285, 298 and309 together distinguish L opsin from M opsin.

The proteins recited (d)-(z) are exemplary eye disease-associated genessuch as in retinitis pigmentosa (polypeptides “e”-“l”, “s”-“y”),incomplete achromatopsia (polypeptide “m”), Stargardt's (polypeptide“d”); Leber congenital amaurosis (polypeptide “z”); cone dystrophy, suchas cone dystrophy type 4 (polypeptide “n”); retinal cone dystrophy ; forexample, retinal cone dystrophy type 3A (polypeptide “o”) ; Cone-roddystrophy (polypeptide “p”); achromatopsia (polypeptide “q′); and totalcolor blindness, for example, among Pingelapese Islanders (polypeptide“r”).

In one embodiment of the invention, the transgene coding sequence ismodified, or “codon optimized” to enhance expression by replacinginfrequently represented codons with more frequently represented codons.The coding sequence is the portion of the mRNA sequence that encodes theamino acids for translation. During translation, each of 61trinucleotide codons are translated to one of 20 amino acids, leading toa degeneracy, or redundancy, in the genetic code. However, differentcell types, and different animal species, utilize tRNAs (each bearing ananticodon) coding for the same amino acids at different frequencies.When a gene sequence contains codons that are infrequently representedby the corresponding tRNA, the ribosome translation machinery may slow,impeding efficient translation. Expression can be improved via “codonoptimization” for a particular species, where the coding sequence isaltered to encode the same protein sequence, but utilizing codons thatare highly represented, and/or utilized by highly expressed humanproteins (Cid-Arregui et al., 2003; J. Virol. 77: 4928). In one aspectof the present invention, the coding sequence of the transgene ismodified to replace codons infrequently expressed in mammal or inprimates with codons frequently expressed in primates. For example, insome embodiments, the coding sequence encoded by the transgene encodes apolypeptide having at least 85% sequence identity to a polypeptideencoded by a sequence disclosed above or herein, for example at least90% sequence identity, e.g. at least 95% sequence identity, at least 98%identity, at least 99% identity, wherein at least one codon of thecoding sequence has a higher tRNA frequency in humans than thecorresponding codon in the sequence disclosed above or herein.

In an additional embodiment of the invention, the transgene codingsequence is modified to enhance expression by termination or removal ofopen reading frames (ORFs) that do not encode the desired transgene. Anopen reading frame (ORF) is the nucleic acid sequence that follows astart codon and does not contains a stop codons. ORFs may be in theforward or reverse orientation, and may be “in frame” or “out of frame”compared with the gene of interest. Such open reading frames have thepotential to be expressed in an expression cassette alongside the geneof interest, and could lead to undesired adverse effects. In one aspectof the present invention, the coding sequence of the transgene has beenmodified to remove open reading frames by further altering codon usage.This was done by eliminating start codons (ATG) and introducing stopcodons (TAG, TAA, or TGA) in reverse orientation or out-of-frame ORFs,while preserving the amino acid sequence and maintaining highly utilizedcodons in the gene of interest (i.e., avoiding codons withfrequency<20%). In the present invention, the transgene coding sequencemay be optimized by either of codon optimization and removal ofnon-transgene ORFs or using both techniques. As will be apparent to oneof ordinary skill in the art, it is preferable to remove or minimizenon-transgene ORFs after codon optimization in order to remove ORFsintroduced during codon optimization. Examples of codon optimization andremoval of ORFs are shown in FIGS. 3A-3C.

In some embodiments, the polynucleotide cassette of the presentinvention further comprises a polyadenylation region. As is understoodin the art, RNA polymerase II transcripts are terminated by cleavage andadditional of a polyadenylation region, also known as a poly A signal,poly A region or poly A tail. The poly A region contains multipleconsecutive adenosine monophosphates, often with repeats of the motifAAUAAA. Several efficient polyadenylation sites have been identified,including those from SV40, bovine growth hormone, human growth hormoneand rabbit beta globin (Xu et al, 2001; Gene 272: 149; Xu et al., 2002;J Control Rel. 81:155). The most efficient polyA signal for expressionof a transgene in cone cells may depend on the cell type and species ofinterest and the particular vector used. In some embodiments of theinvention, the polynucleotide cassette comprises, consists essentiallyof, or consists of the polyA region selected from the group consistingof SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,SEQ ID NO:90 or SEQ ID NO:91 or functional fragment or variant thereofof any of the preceding sequences. In certain embodiments, the polyAregion comprises SEQ ID NO:90 or a variant thereof. In some suchembodiments, the polyA region consists essentially of SEQ ID NO:90 or avariant thereof.

As will be appreciated by the ordinarily skilled artisan, two or more ofthe aforementioned polynucleotide elements may be combined to create thepolynucleotide cassettes of the present disclosure. Thus, for example,the subject polynucleotide cassette may comprise a promoter regioncomprising an improved promoter sequence in operable linkage with animproved 5′UTR sequence, for example SEQ ID NO:80 in operablecombination with SEQ ID NO:84 or SEQ ID NO:85, see, e.g. SEQ ID NO:2,SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:93, SEQ ID NO:94, or SEQ ID NO:95.As another example, the subject polynucleotide cassette may comprise animproved enhancer sequence or region in operable linkage with animproved promoter sequence or region, for example SEQ ID NO:51 or SEQ IDNO:52 in operable combination with SEQ ID NO:80, SEQ ID NO:2, SEQ IDNO:54, SEQ ID NO:55, or SEQ ID NO:93; see, e.g. SEQ ID NO:92 or SEQ IDNO:95. As another example, the subject polynucleotide cassette maycomprise an improved 5′UTR sequence in operable linkage with an improvedintron sequence, for example SEQ ID NO:84 or SEQ ID NO:86 in operablecombination with SEQ ID NO:60; see, e.g. SEQ ID NO:94 or SEQ ID NO:95.As another example, the subject polynucleotide cassette may comprise animproved 5′UTR sequence in operable linkage with an improved intronsequence and an improved Kozak sequence, for example, SEQ ID NO:84 orSEQ ID NO:86 in operable combination with SEQ ID NO:60 and with SEQ IDNO:73; see, e.g. SEQ ID NO:95. As another example, the subjectpolynucleotide cassette may comprise an improved enhancer, improvedpromoter, improved 5′UTR, improved intron, improved kozak and improvedpolyA region in operable linkage; see, e.g. SEQ ID NO:95. Othercombinations of elements both as disclosed herein or as known in the artwill be readily appreciated by the ordinarily skilled artisan.

Additionally, as will be recognized by one of ordinary skill in the art,the polynucleotide cassettes may optionally contain other elementsincluding, but not limited to restriction sites to facilitate cloningand regulatory elements for a particular gene expression vector.Examples of regulatory sequence include ITRs for AAV vectors, bacterialsequences for plasmid vectors, attP or attB sites for phage integrasevectors, and transposable elements for transposons.

Gene Therapy Vectors

As alluded to above, in some aspects of the present invention, thesubject polynucleotide cassettes are used to deliver a gene to conecells of an animal, e.g. to determine the effect that the gene has oncell viability and/or function, to treat a cone cell disorder, etc.Accordingly, in some aspects of the invention, the composition thatprovides for the expression of a transgene in cone cells is a genedelivery vector, wherein the gene delivery vector comprises thepolynucleotide cassettes of the present disclosure.

Any convenient gene therapy vector that finds use deliveringpolynucleotide sequences to cone cells is encompassed by the genedelivery vectors of the present disclosure. For example, the vector maycomprise single or double stranded nucleic acid, e.g. single stranded ordouble stranded DNA. For example, the gene delivery vector may be anaked DNA, e.g. a plasmid, a minicircle, etc. As another example, thegene delivery vector may be a virus, e.g. an adenovirus, anadeno-associated virus, or a retrovirus, e.g. Moloney murine leukemiavirus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon apeleukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friendmurine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous SarcomaVirus (RSV)) or lentivirus. While embodiments encompassing the use ofadeno-associated virus are described in greater detail below, it isexpected that the ordinarily skilled artisan will appreciate thatsimilar knowledge and skill in the art can be brought to bear on non-AAVgene therapy vectors as well. See, for example, the discussion ofretroviral vectors in, e.g., U.S. Pat. No. 7,585,676 and U.S. Pat. No.8,900,858, and the discussion of adenoviral vectors in, e.g. U.S. Pat.No. 7,858,367, the full disclosures of which are incorporated herein byreference.

In some embodiments, the gene delivery vector is a recombinantadeno-associated virus (rAAV). In such embodiments, the subjectpolynucleotide cassette is flanked on the 5′ and 3′ ends by functionalAAV inverted terminal repeat (ITR) sequences. By “functional AAV ITRsequences” is meant that the ITR sequences function as intended for therescue, replication and packaging of the AAV virion. Hence, AAV ITRs foruse in the gene delivery vectors of the invention need not have awild-type nucleotide sequence, and may be altered by the insertion,deletion or substitution of nucleotides or the AAV ITRs may be derivedfrom any of several AAV serotypes, e.g. AAV1, AAV2, AAV3, AAV4, AAVS,AAV6, AAV7, AAV8, AAV9, AAV10. Preferred AAV vectors have the wild typeREP and CAP genes deleted in whole or part, but retain functionalflanking ITR sequences.

In such embodiments, the subject polynucleotide cassette is encapsidatedwithin an AAV capsid, which may be derived from any adeno-associatedvirus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4,AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV capsidmay be a wild type, or native, capsid. Wild type AAV capsids ofparticular interest include AAV2, AAVS, and AAV9. However, as with theITRs, the capsid need not have a wild-type nucleotide sequence, butrather may be altered by the insertion, deletion or substitution ofnucleotides in the VP1, VP2 or VP3 sequence, so long as the capsid isable to transduce cone cells. Put another way, the AAV capsid may be avariant AAV capsid. Variant AAV capsids of particular interest includethose comprising a peptide insertion within residues 580-600 of AAV2 orthe corresponding residues in another AAV, e.g. LGETTRP, NETITRP,KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, orSTGKVPN, as disclosed inUS Application No. US 2014/0294771, the full disclosure of which isincorporated by reference herein. In some embodiments, the AAV vector isa “pseudotyped” AAV created by using the capsid (cap) gene of one AAVand the rep gene and ITRs from a different AAV, e.g. a pseudotyped AAV2created by using rep from AAV2 and cap from AAV1, AAV3, AAV4, AAVS,AAV6, AAV7, AAV8, or AAV9 together with a plasmid containing a vectorbased on AAV2. For example, the AAV vector may be rAAV2/1, rAAV2/3,rAAV2/4, rAAV2/5, rAAV2/6, rAAV2/7, rAAV2/8, rAAV2/9, etc. Preferably,the rAAV is replication defective, in that the AAV vector cannotindependently further replicate and package its genome. For example,when cone cells are transduced with rAAV virions, the gene is expressedin the transduced cone cells, however, due to the fact that thetransduced cone cells lack AAV rep and cap genes and accessory functiongenes, the rAAV is not able to replicate.

Gene therapy vectors, e.g. rAAV) virions encapsulating thepolynucleotide cassettes of the present disclosure, may be producedusing standard methodology. For example, in the case of rAAV virions, anAAV expression vector according to the invention may be introduced intoa producer cell, followed by introduction of an AAV helper construct,where the helper construct includes AAV coding regions capable of beingexpressed in the producer cell and which complement AAV helper functionsabsent in the AAV vector. This is followed by introduction of helpervirus and/or additional vectors into the producer cell, wherein thehelper virus and/or additional vectors provide accessory functionscapable of supporting efficient rAAV virus production. The producercells are then cultured to produce rAAV. These steps are carried outusing standard methodology. Replication-defective AAV virionsencapsulating the recombinant AAV vectors of the instant invention aremade by standard techniques known in the art using AAV packaging cellsand packaging technology. Examples of these methods may be found, forexample, in U.S. Pat. Nos. 5,436,146; 5,753,500, 6,040,183, 6,093,570and 6,548,286, expressly incorporated by reference herein in theirentirety. Further compositions and methods for packaging are describedin Wang et al. (US 2002/0168342), also incorporated by reference hereinin its entirety.

Any suitable method for producing viral particles for delivery of thesubject polynucleotide cassettes can be used, including but not limitedto those described in the examples that follow. Any concentration ofviral particles suitable to effectively transducer cone cells can beprepared for contacting cone cells in vitro or in vivo. For example, theviral particles may be formulated at a concentration of 10⁸ vectorgenomes per ml or more, for example, 5×10⁸ vector genomes per mL; 10⁹vector genomes per mL; 5×10⁹ vector genomes per mL, 10¹⁰ vector genomesper mL, 5×10¹⁰ vector genomes per mL; 10¹¹ vector genomes per mL; 5×10¹¹vector genomes per mL; 10¹² vector genomes per mL; 5×10¹² vector genomesper mL; 10¹³ vector genomes per mL; 1.5×10¹³ vector genomes per mL;3×10¹³ vector genomes per mL; 5×10¹³ vector genomes per mL; 7.5×10¹³vector genomes per mL; 9×10¹³ vector genomes per mL; 1×10¹⁴ vectorgenomes per mL, 5×10¹⁴ vector genomes per mL or more, but typically notmore than 1×10¹⁵ vector genomes per mL. Similarly, any total number ofviral particles suitable to provide appropriate transduction of retinalcone cells to confer the desired effect or treat the disease can beadministered to the mammal or to the primate's eye. In various preferredembodiments, at least 10⁸; 5×10⁸; 10⁹; 5×10⁹, 10¹⁰, 5×10¹⁰; 10¹¹;5×10¹¹; 10¹²; 5×10¹²; 10¹³; 1.5×10¹³; 3×10¹³; 5×10¹³; 7.5×10¹³; 9×10¹³,1×10¹⁴ viral particles, or 5×10¹⁴ viral particles or more, but typicallynot more than 1×10¹⁵ viral particles are injected per eye. Any suitablenumber of administrations of the vector to the mammal or the primate eyecan be made. In one embodiment, the methods comprise a singleadministration; in other embodiments, multiple administrations are madeover time as deemed appropriate by an attending clinician.

The subject viral vector may be formulated into any suitable unitdosage, including, without limitation, 1×10⁸ vector genomes or more, forexample, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ vector genomes ormore, in certain instances, 1×10¹⁴ vector genomes, but usually no morethan 4×10¹⁵ vector genomes. In some cases, the unit dosage is at mostabout 5×10¹⁵ vector genomes, e.g. 1×10¹⁴ vector genomes or less, forexample 1×10¹³, 1×10¹², 1×10¹¹, 1×10¹⁰, or 1×10⁹ vector genomes or less,in certain instances 1×10⁸ vector genomes or less, and typically no lessthan 1×10⁸ vector genomes. In some cases, the unit dosage is 1×10¹⁰ to1×10¹¹ vector genomes. In some cases, the unit dosage is 1×10¹⁰ to3×10¹² vector genomes. In some cases, the unit dosage is 1×10⁹ to 3×10¹³vector genomes. In some cases, the unit dosage is 1×10⁸ to 3×10¹⁴ vectorgenomes.

In some cases, the unit dosage of pharmaceutical composition may bemeasured using multiplicity of infection (MOI). By MOI it is meant theratio, or multiple, of vector or viral genomes to the cells to which thenucleic acid may be delivered. In some cases, the MOI may be 1×10⁶. Insome cases, the MOI may be 1×10⁵-1×10⁷. In some cases, the MOI may be1×10⁴-1×10⁸. In some cases, recombinant viruses of the disclosure are atleast about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ MOI. In some cases, recombinant viruses of this disclosureare 1×10⁸ to 3×10¹⁴ MOI. In some cases, recombinant viruses of thedisclosure are at most about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵,1×10¹⁶, 1×10¹⁷ and 1×10¹⁸ MOI.

In some aspects, the amount of pharmaceutical composition comprisesabout 1×10⁸ to about 1×10¹⁵ recombinant viruses, about 1×10⁹ to about1×10¹⁴ recombinant viruses, about 1×10¹⁰ to about 1×10¹³ recombinantviruses, or about 1×10¹¹ to about 3×10¹² recombinant viruses.

In preparing the subject rAAV compositions, any host cells for producingrAAV virions may be employed, including, for example, mammalian cells(e.g. 293 cells), insect cells (e.g. SF9 cells), microorganisms andyeast. Host cells can also be packaging cells in which the AAV rep andcap genes are stably maintained in the host cell or producer cells inwhich the AAV vector genome is stably maintained and packaged. Exemplarypackaging and producer cells are derived from SF-9, 293, A549 or HeLacells. AAV vectors are purified and formulated using standard techniquesknown in the art.

For instances in which cone cells are to be contacted in vivo, thesubject polynucleotide cassettes or gene delivery vectors comprising thesubject polynucleotide cassette can be treated as appropriate fordelivery to the eye. In particular, the present invention includepharmaceutical compositions comprising a polynucleotide cassetee or genedelivery vector described herein and a pharmaceutically-acceptablecarrier, diluent or excipient. The subject polynucleotide cassettes orgene delivery vector can be combined with pharmaceutically-acceptablecarriers, diluents and reagents useful in preparing a formulation thatis generally safe, non-toxic, and desirable, and includes excipientsthat are acceptable for primate use. Such excipients can be solid,liquid, semisolid, or, in the case of an aerosol composition, gaseous.Examples of such carriers or diluents include, but are not limited to,water, saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Supplementary active compounds can also be incorporated intothe formulations. Solutions or suspensions used for the formulations caninclude a sterile diluent such as water for injection, saline solution,fixed oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial compounds such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates; detergents such asTween 20 to prevent aggregation; and compounds for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide.

Pharmaceutical compositions suitable for internal use in the presentinvention further include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In somecases, the composition issterile and should be fluid to the extent that easy syringabilityexists. In certain embodiments, it is stable under the conditions ofmanufacture and storage and is preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can be,e.g., a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the internal compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In one embodiment, active compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser, e.g. syringe, e.g. a prefilled syringe, together withinstructions for administration.

The pharmaceutical compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal comprising ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bio-equivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions.

The term “pharmaceutically acceptable salt” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Avariety of pharmaceutically acceptable salts are known in the art anddescribed, e.g., in in “Remington's Pharmaceutical Sciences”, 17thedition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa.,USA, 1985 (and more recent editions thereof), in the “Encyclopaedia ofPharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), InformaHealthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2(1977). Also, for a review on suitable salts, see Handbook ofPharmaceutical Salts: Properties, Selection, and Use by Stahl andWermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like Amines comprise N-N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

The subject polynucleotide cassette or gene delivery vector,e.g.recombinant virus (virions), can be incorporated into pharmaceuticalcompositions for administration to mammalian patients, particularlyprimates and more particularly humans. The subject polynucleotidecassette or gene delivery vector, e.g. virions can be formulated innontoxic, inert, pharmaceutically acceptable aqueous carriers,preferably at a pH ranging from 3 to 8, more preferably ranging from 6to 8. Such sterile compositions will comprise the vector or virioncontaining the nucleic acid encoding the therapeutic molecule dissolvedin an aqueous buffer having an acceptable pH upon reconstitution.

In some embodiments, the pharmaceutical composition provided hereincomprise a therapeutically effective amount of a vector or virion inadmixture with a pharmaceutically acceptable carrier and/or excipient,for example saline, phosphate buffered saline, phosphate and aminoacids, polymers, polyols, sugar, buffers, preservatives and otherproteins. Exemplary amino acids, polymers and sugars and the like areoctylphenoxy polyethoxy ethanol compounds, polyethylene glycolmonostearate compounds, polyoxyethylene sorbitan fatty acid esters,sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran,sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine orhuman serum albumin, citrate, acetate, Ringer's and Hank's solutions,cysteine, arginine, carnitine, alanine, glycine, lysine, valine,leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, thisformulation is stable for at least six months at 4° C.

In some embodiments, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the bufferin which the pharmaceutical composition comprising the tumor suppressorgene contained in the adenoviral vector delivery system, may be in therange of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to7.4.

Methods

As alluded to above, the subject polynucleotide cassettes and genedelivery vectors, referred to collectively herein as the “subjectcompositions”, find use in expressing a transgene in cone cells of ananimal. For example, the subject compositions may be used in research,e.g. to determine the effect that the gene has on cone cell viabilityand/or function. As another example, the subject compositions may beused in medicine, e.g. to treat a cone cell disorder. Thus, in someaspects of the invention, methods are provided for the expression of agene in cone cells, the method comprising contacting cone cells with acomposition of the present disclosure. In some embodiments, contactingoccurs in vitro. In some embodiments, contacting occurs in vivo, i.e.,the subject composition is administered to a subject.

For instances in which cone cells are to be contacted in vitro with asubject polynucleotide cassette or gene delivery vector comprising asubject polynucleotide cassette, the cells may be from any mammalianspecies, e.g. rodent (e.g. mice, rats, gerbils, squirrels), rabbit,feline, canine, goat, ovine, pig, equine, bovine, primate, human. Cellsmay be from established cell lines, e.g. WERI cells, 661W cells, or theymay be primary cells, where “primary cells”, “primary cell lines”, and“primary cultures” are used interchangeably herein to refer to cells andcells cultures that have been derived from a subject and allowed to growin vitro for a limited number of passages, i.e. splittings, of theculture. For example, primary cultures are cultures that may have beenpassaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15times, but not enough times go through the crisis stage. Typically, theprimary cell lines of the present invention are maintained for fewerthan 10 passages in vitro.

If the cells are primary cells, they may be harvested from a mammal byany convenient method, e.g. whole explant, biopsy, etc. An appropriatesolution may be used for dispersion or suspension of the harvestedcells. Such solution will generally be a balanced salt solution, e.g.normal saline, PBS, Hank's balanced salt solution, etc., convenientlysupplemented with fetal calf serum or other naturally occurring factors,in conjunction with an acceptable buffer at low concentration, generallyfrom 5-25 mM. Convenient buffers include HEPES, phosphate buffers,lactate buffers, etc. The cells may be used immediately, or they may bestored, frozen, for long periods of time, being thawed and capable ofbeing reused. In such cases, the cells will usually be frozen in 10%DMSO, 50% serum, 40% buffered medium, or some other such solution as iscommonly used in the art to preserve cells at such freezingtemperatures, and thawed in a manner as commonly known in the art forthawing frozen cultured cells.

To promote expression of the transgene, the subject polynucleotidecassette or gene delivery vector comprising a subject polynucleotidecassette will be contacted with the cells for about 30 minutes to 24hours or more, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours,18 hours, 20 hours, 24 hours, etc. The subject polynucleotide cassetteor gene delivery vector comprising a subject polynucleotide cassette maybe provided to the subject cells one or more times, e.g. one time,twice, three times, or more than three times, and the cells allowed toincubate with the agent(s) for some amount of time following eachcontacting event e.g. 16-24 hours, after which time the media isreplaced with fresh media and the cells are cultured further. Contactingthe cells may occur in any culture media and under any cultureconditions that promote the survival of the cells. For example, cellsmay be suspended in any appropriate nutrient medium that is convenient,such as Iscove's modified DMEM or RPMI 1640, supplemented with fetalcalf serum or heat inactivated goat serum (about 5-10%), L-glutamine, athiol, particularly 2-mercaptoethanol, and antibiotics, e.g. penicillinand streptomycin. The culture may contain growth factors to which thecells are responsive. Growth factors, as defined herein, are moleculescapable of promoting survival, growth and/or differentiation of cells,either in culture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors.

Typically, an effective amount of subject polynucleotide cassette orgene delivery vector comprising a subject polynucleotide cassette isprovided to produce the expression of the transgene in cells. Asdiscussed elsewhere herein, the effective amount may be readilydetermined empirically, e.g. by detecting the presence or levels oftransgene gene product, by detecting an effect on the viability orfunction of the cone cells, etc. Typically, an effect amount of subjectpolynucleotide cassette or gene delivery vector comprising a subjectpolynucleotide cassette will promote greater expression of the transgenein cone cells than the same amount of a polynucleotide cassette as knownin the art, e.g. a pR2.1 (nucleotides 1-2274 of SEQ ID NO:50), pR1.7,pR1.5, pR1.1, or IRBP/GNAT2 cassette. Typically, expression will beenhanced 2-fold or more relative to the expression from a reference, orcontrol, polynucleotide cassette e.g. as known in the art, for example3-fold, 4-fold, or 5-fold or more, in some instances 10-fold, 20-fold or50-fold or more, e.g. 100-fold.

In some embodiments, as when the transgene is a selectable marker, thepopulation of cells may be enriched for those comprising the subjectpolynucleotide cassette by separating the modified cells from theremaining population. Separation may be by any convenient separationtechnique appropriate for the selectable marker used. For example, ifthe transgene is a fluorescent marker, cells may be separated byfluorescence activated cell sorting, whereas if the transgene is a cellsurface marker, cells may be separated from the heterogeneous populationby affinity separation techniques, e.g. magnetic separation, affinitychromatography, “panning” with an affinity reagent attached to a solidmatrix, or other convenient technique. Techniques providing accurateseparation include fluorescence activated cell sorters, which can havevarying degrees of sophistication, such as multiple color channels, lowangle and obtuse light scattering detecting channels, impedancechannels, etc. The cells may be selected against dead cells by employingdyes associated with dead cells (e.g. propidium iodide). Any techniquemay be employed which is not unduly detrimental to the viability of thecells. Cell compositions that are highly enriched for cells comprisingthe subject polynucleoties are achieved in this manner. By “highlyenriched”, it is meant that the genetically modified cells will be 70%or more, 75% or more, 80% or more, 85% or more, 90% or more of the cellcomposition, for example, about 95% or more, or 98% or more of the cellcomposition. In other words, the composition may be a substantially purecomposition of genetically modified cells.

For instances in which cone cells are to be contacted in vivo with asubject polynucleotide cassette or gene delivery vector comprising asubject polynucleotide cassette, the subject may be any mammal, e.g.rodent (e.g. mice, rats, gerbils), rabbit, feline, canine, goat, ovine,pig, equine, bovine, or primate. In certain embodiments, the subject isa primate of the Parvorder Catarrhini. As is known in the art,Catarrhini is one of the two subdivisions of the higher primates (theother being the New World monkeys), and includes Old World monkeys andthe apes, which in turn are further divided into the lesser apes orgibbons and the great apes, consisting of the orangutans, gorillas,chimpanzees, bonobos, and humans. In a further preferred embodiment, theprimate is a human.

The subject composition may be administered to the retina of the subjectby any suitable method. For example, the subject composition may beadministered intraocularly via intravitreal injection or subretinalinjection. The general methods for delivering a vector via intravitrealinjection or via subretinal injection may be illustrated by thefollowing brief outlines. These examples are merely meant to illustratecertain features of the methods, and are in no way meant to be limiting.

For subretinal administration, the vector can be delivered in the formof a suspension injected subretinally under direct observation using anoperating microscope. Typically, a volume of 1 to 200 uL, e.g. 50 uL,100 uL, 150 ul, or 200 uL, but usually no more than 200 uL, of thesubject composition will be administered by such methods. This proceduremay involve vitrectomy followed by injection of vector suspension usinga fine cannula through one or more small retinotomies into thesubretinal space. Briefly, an infusion cannula can be sutured in placeto maintain a normal globe volume by infusion (of e.g. saline)throughout the operation. A vitrectomy is performed using a cannula ofappropriate bore size (for example 20 to 27 gauge), wherein the volumeof vitreous gel that is removed is replaced by infusion of saline orother isotonic solution from the infusion cannula. The vitrectomy isadvantageously performed because (1) the removal of its cortex (theposterior hyaloid membrane) facilitates penetration of the retina by thecannula; (2) its removal and replacement with fluid (e.g. saline)creates space to accommodate the intraocular injection of vector, and(3) its controlled removal reduces the possibility of retinal tears andunplanned retinal detachment.

For intravitreal administration, the vector can be delivered in the formof a suspension. Initially, topical anesthetic is applied to the surfaceof the eye followed by a topical antiseptic solution. The eye is heldopen, with or without instrumentation, and the vector is injectedthrough the sclera with a short, narrow, for example a 30 gauge needle,into the vitreous cavity of the eye of a subject under directobservation. Typically, a volume of 1 to 100 uL, e.g. 25 uL, 50 uL, or100 uL, and usually no more than 100uL, of the subject composition maybe delivered to the eye by intravitreal injection without removing thevitreous. Alternatively, a vitrectomy may be performed, and the entirevolume of vitreous gel is replaced by an infusion of the subjectcomposition. In such cases, up to about 4 mL of the subject compositionmay be delivered, e.g. to a human eye. Intravitreal administration isgenerally well tolerated. At the conclusion of the procedure, there issometimes mild redness at the injection site. There is occasionaltenderness, but most patients do not report any pain. No eye patch oreye shield is necessary after this procedure, and activities are notrestricted. Sometimes, an antibiotic eye drop is prescribed for severaldays to help prevent infection.

The methods and compositions of the present disclosure find use in thetreatment of any condition that can be addressed, at least in part, bygene therapy of cone photoreceptor cells. Thus, the compositions andmethods of the present disclosure find use in the treatment ofindividuals in need of a cone cell therapy. By a person in need of acone cell therapy, it is meant an individual having or at risk ofdeveloping a cone cell disorder. By a “cone cell disorder” it is meantany disorder impacting retinal cone cells, including but not limited tovision disorders of the eye that are associated with a defect withincone cells, i.e. a cone-instrinsic defect, e.g. macular dystrophies suchas Stargardt's macular dystrophy, cone dystrophy, cone-rod dystrophy,Spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1; as well ascolor vision disorders, including achromatopsia, incompleteachromatopsia, blue cone monochromacy, and protan, deutan, and tritandefects; as well as vision disorders of the central macula (withinprimates) that may be treated by targeting cone cells, e.g. age-relatedmacular degeneration, macular telangiectasia, retinitis pigmentosa,diabetic retinopathy, retinal vein occlusions, glaucoma, Sorsby's fundusdystrophy, adult vitelliform macular dystrophy, Best's disease, rod-conedystrophy, Leber's congenital amaurosis, and X-linked retinoschisis.

Stargardt's macular dystrophy. Stargardt's macular dystrophy, also knownas Stargardt Disease and fundus flavimaculatus, is an inherited form ofjuvenile macular degeneration that causes progressive vision lossusually to the point of legal blindness. The onset of symptoms usuallyappears between the ages of six and thirty years old (average of about16-18 years). Mutations in several genes, including ABCA4, CNGB3,ELOVL4, PROM1, are associated with the disorder. Symptoms typicallydevelop by twenty years of age, and include wavy vision, blind spots,blurriness, impaired color vision, and difficulty adapting to dimlighting. The main symptom of Stargardt disease is loss of visualacuity, which ranges from 20/50 to 20/200. In addition, those withStargardt disease are sensitive to glare; overcast days offer somerelief. Vision is most noticeably impaired when the macula is damaged,which can be observed by fundus exam.

Cone dystrophy. Cone dystrophy (COD) is an inherited ocular disordercharacterized by the loss of cone cells. The most common symptoms ofcone dystrophy are vision loss (age of onset ranging from the late teensto the sixties), sensitivity to bright lights, and poor color vision.Visual acuity usually deteriorates gradually, but it can deterioraterapidly to 20/200; later, in more severe cases, it drops to “countingfingers” vision. Color vision testing using color test plates (HRRseries) reveals many errors on both red-green and blue-yellow plates. Itis believed that the dystrophy is primary, since subjective andobjective abnormalities of cone function are found beforeophthalmoscopic changes can be seen. However, the retinal pigmentepithelium (RPE) rapidly becomes involved, leading to a retinaldystrophy primarily involving the macula. The fundus exam viaophthalmoscope is essentially normal early on in cone dystrophy, anddefinite macular changes usually occur well after visual loss. The mostcommon type of macular lesion seen during ophthalmoscopic examinationhas a bull's-eye appearance and consists of a doughnut-like zone ofatrophic pigment epithelium surrounding a central darker area. Inanother, less frequent form of cone dystrophy there is rather diffuseatrophy of the posterior pole with spotty pigment clumping in themacular area. Rarely, atrophy of the choriocapillaris and largerchoroidal vessels is seen in patients at an early stage. Fluoresceinangiography (FA) is a useful adjunct in the workup of someone suspectedto have cone dystrophy, as it may detect early changes in the retinathat are too subtle to be seen by ophthalmoscope. Because of the widespectrum of fundus changes and the difficulty in making the diagnosis inthe early stages, electroretinography (ERG) remains the best test formaking the diagnosis. Abnormal cone function on the ERG is indicated bya reduced single-flash and flicker response when the test is carried outin a well-lit room (photopic ERG). Mutations in several genes, includingGUCA1A, PDE6C, PDE6H, and RPGR, are associated with the disorder.

Cone-rod dystrophy. Cone-rod dystrophy (CRD, or CORD) is an inheritedretinal dystrophy that belongs to the group of pigmentary retinopathies.CRD is characterized by retinal pigment deposits visible on fundusexamination, predominantly localized to the macular region and the lossof both cone and rod cells. In contrast to rod-cone dystrophy (RCD)resulting from the primary loss in rod photoreceptors and later followedby the secondary loss in cone photoreceptors, CRD reflects the oppositesequence of events: primary cone involvement, or, sometimes, byconcomitant loss of both cones and rods. Symptoms include decreasedvisual acuity, color vision defects, photoaversion and decreasedsensitivity in the central visual field, later followed by progressiveloss in peripheral vision and night blindness. Mutations in severalgenes, including ADAM9, PCDH21, CRX, GUCY2D, PITPNM3, PROM1, PRPH2,RAX2, RIMS1, RPGR, and RPGRIP1, are associated with the disorder.

Spinocerebellar ataxia type 7. Spinocerebellar ataxia is a progressive,degenerative, inherited disease characterized by slowly progressiveincoordination of gait and is often associated with poor coordination ofhands, speech, and eye movements. There are multiple types of SCA, withSpinocerebellar ataxia type 7 (SCA-7) differing from most other SCAs inthat visual problems can occur in addition to poor coordination. SCA-7is associated with automosmal dominant mutations in the ATXN7/SCA7 gene.When the disease manifests itself before age 40, visual problems ratherthan poor coordination are typically the earliest signs of disease.Early symptoms include difficulty distinguishing colors and decreasedcentral vison. In addition, symptoms of ataxia (incoordination, slow eyemovements, and mild changes in sensation or reflexes) may be detectable.Loss of motor control, unclear speech, and difficulty swallowing becomeprominent as the disease progresses.

Bardet-Biedl syndrome-1. Bardet-Biedl syndrome-1 (BBS-1) is apleiotropic disorder with variable expressivity and a wide range ofclinical variability observed both within and between families. The mainclinical features are rod-cone dystrophy, with childhood-onset visualloss preceded by night blindness; postaxial polydactyly; truncal obesitythat manifests during infancy and remains problematic throughoutadulthood; specific learning difficulties in some but not allindividuals; male hypogenitalism and complex female genitourinarymalformations; and renal dysfunction, a major cause of morbidity andmortality. Vision loss is one of the major features of Bardet-Biedlsyndrome. Problems with night vision become apparent by mid-childhood,followed by blind spots that develop in the peripheral vision. Overtime, these blind spots enlarge and merge to produce tunnel vision. Mostpeople with Bardet-Biedl syndrome also develop blurred central vision(poor visual acuity) and become legally blind by adolescence or earlyadulthood. Bardet-Biedl syndrome can result from mutations in at least14 different genes (often called BBS genes) known or suspected to playcritical roles in cilia function, with mutations in BBS1 and BBS10 beingthe most common.

Achromatopsia. Achromatopsia, or Rod monochromatism, is a disorder inwhich subjects experience a complete lack of the perception of color,such that the subject sees only in black, white, and shades of grey.Other symptoms include reduced visual acuity, photophobia, nystagmus,small central scotoma, and eccentric fixation. The disorder isfrequently noticed first in children around six months of age by theirphotophobic activity and/or their nystagmus. Visual acuity and stabilityof the eye motions generally improve during the first 6-7 years of life(but remain near 20/200). Mutations in CNGB3, CNGA3, GNAT2, PDE6C, andPDE6HI have been associated with the disorder.

Incomplete achromatopsia. Incomplete achromatopsia is similar toAchromatopsia but with less penetrance. In incomplete achromatopsia, thesymptoms are similar to those of complete achromatopsia except in adiminished form. Individuals with incomplete achromatopsia have reducedvisual acuity with or without nystagmus or photophobia. Furthermore,these individuals show only partial impairment of cone cell function butagain have retained rod cell function.

Blue cone monochromacy. Blue cone (S cone) monochromatism (BCM) is arare X-linked congenital stationary cone dysfunction syndrome, affectingapproximately 1 in 100,000 individuals. Affected males with BCM have nofunctional long wavelength sensitive (L) or medium wavelength sensitive(M) cones in the retina, due to mutations at the genetic locus for the Land M-opsin genes. Color discrimination is severely impaired from birth,and vision is derived from the remaining preserved S cones and rodphotoreceptors. BCM typically presents with reduced visual acuity (6/24to 6/60), pendular nystagmus, photophobia, and patients often havemyopia. The rod-specific and maximal electroretinogram (ERG) usuallyshow no definite abnormality, whereas the 30 Hz cone ERG cannot bedetected. Single flash photopic ERG is often recordable, albeit smalland late, and the S cone ERG is well preserved.

Color vision deficiency. Color vision deficiency (CVD), or colorblindness, is the inability or decreased ability to see color, orperceive color differences, under normal lighting conditions.Individuals suffering from color blindness may be identified as suchusing any of a number of color vision tests, e.g., color ERG (cERG),pseudoisochromatic plates (Ishihara plates, Hardy-Rand-Ritterpolychromatic plates), the Farnsworth-Munsell 100 hue test, theFarnsworth's panel D-15, the City University test, Kollner's rule, etc.Examples of color vision deficiencies include protan defects, deutandefects, and tritan defects. Protan defects include protanopia (aninsensitivity to red light) and protanomaly (a reduced sensitivity tored light), and are associated with mutations in the L-Opsin gene(OPN1LW). Deutan defects include deuteranopia (an insensitivity to greenlight) and deutanomaly (a reduced sensitivity to green light), and areassociated with mutations in the M-Opsin gene (OPN1MW). Tritan defectsinclude tritanopia (an insensitivity to blue light) and tritanomaly (areduced sensitivity to blue light), and are associated with mutations inthe S-Opsin gene (OPN1SW).

Age-related macular degeneration. Age-related macular degeneration (AMD)is one of the leading causes of vision loss in people over the age of 50years. AMD mainly affects central vision, which is needed for detailedtasks such as reading, driving, and recognizing faces. The vision lossin this condition results from a gradual deterioration of photoreceptorsin the macula. Side (peripheral) vision and night vision are generallynot affected.

Researchers have described two major types of age-related maculardegeneration, known as the dry, or “nonexudative” form, and the wet, or“exudative” or “neovascular”, form, both of which may be treated bydelivering transgenes in the context of the subject polynucleotidecassettes.

Dry AMD is characterized by a buildup of yellow deposits called drusenbetween the retinal pigment epithelium and the underlying choroid of themacula, which may be observed by Fundus photography. This results in aslowly progressive loss of vision. The condition typically affectsvision in both eyes, although vision loss often occurs in one eye beforethe other. Other changes may include pigment changes and RPE atrophy.For example, in certain cases called central geographic atrophy, or“GA”, atrophy of the retinal pigment epithelial and subsequent loss ofphotoreceptors in the central part of the eye is observed. Dry AMD hasbeen associated with mutations in CD59 and genes in the complementcascade.

Wet AMD is a progressed state of dry AMD, and occurs in abut 10% of dryAMD patients. Pathological changes include retinal pigment epithelialcells (RPE) dysfunction, fluid collecting under the RPE, and choroidalneovascularization (CNV) in the macular area. Fluid leakage, RPE orneural retinal detachment and bleeding from ruptured blood vessels canoccur in severe cases. Symptoms of wet AMD may include visualdistortions, such as straight lines appearing wavy or crooked, a doorwayor street sign looking lopsided, or objects appearing smaller or fartheraway than they really are; decreased central vision; decreased intensityor brightness of colors; and well-defined blurry spot or blind spot inthe field of vision. Onset may be abrupt and worsen rapidly. Diagnosismay include the use of an Amsler grid to test for defects in thesubject's central vision (macular degeneration may cause the straightlines in the grid to appear faded, broken or distorted), fluoresceinangiogram to observe blood vessel or retinal abnormalities, and opticalcoherence tomography to detect retina swelling or leaking blood vessels.A number of cellular factors have been implicated in the generation ofCNV, among which are vascular endothelial growth factor (VEGF),platelet-derived growth factor (PDGF), pigment epithelium-derived factor(PEDF), hypoxia inducible factor (HIF), angiopoietin (Ang), and othercytokines, mitogen-activated protein kinases (MAPK) and others.

Macular telangiectasia. Macular telangiectasia (MacTel) is a form ofpathologically dilated blood vessels (telangiectasia) in the parafovealregion of the macula. The tissue deteriorates and the retinal structurebecomes scarred due to the development of liquid-filled cysts, whichimpairs nutrition of the photoreceptor cells and destroys visionpermanently. There are two types of MacTel, type 1 and type 2. Maculartelangiectasia type 2 is a bilateral disease, whose prevalence hasrecently been shown to be as high as 0.1% in persons 40 years and older.Biomicroscopy may show reduced retinal transparency, crystallinedeposits, mildly ectatic capillaries, blunted venules, retinal pigmentplaques, foveal atrophy, and neovascular complexes. Fluoresceinangiography shows telangiectatic capillaries predominantly temporal tothe foveola in the early phase and a diffuse hyperfluorescence in thelate phase. High-resolution optical coherence tomography (OCT) mayreveal disruption of the photoreceptor inner segment-outer segmentborder, hyporeflective cavities at the level of the inner or outerretina, and atrophy of the retina in later stages. In Type 1 maculartelangiectasia, the disease almost always occurs in one eye, whichdifferentiates it from Type 2. While MacTel does not usually cause totalblindness, it commonly causes loss of the central vision, which isrequired for reading and driving vision, over a period of 10-20 years.

Retinitis pigmentosa. Retinitis Pigmentosa (RP) is a group of inheriteddisorders characterized by progressive peripheral vision loss and nightvision difficulties (nyctalopia) that can lead to central vision loss.Presenting signs and symptoms of RP vary, but the classic ones includenyctalopia (night blindness, most commonly the earliest symptom in RP);visual loss (usually peripheral, but in advanced cases, central visualloss); and photopsia (seeing flashes of light). Because RP is acollection of many inherited diseases, significant variability exists inthe physical findings. Ocular examination involves assessment of visualacuity and pupillary reaction, as well as anterior segment, retinal, andfunduscopic evaluation. In some instances, the RP is one aspect of asyndrome, e.g. syndromes that are also associated with hearing loss(Usher syndrome, Waardenburg syndrome, Alport syndrome, Refsum disease);Kearns-Sayre syndrome (external ophthalmoplegia, lid ptosis, heartblock, and pigmentary retinopathy); Abetalipoproteinemia (Fatmalabsorption, fat-soluble vitamin deficiencies, spinocerebellardegeneration, and pigmentary retinal degeneration);mucopolysaccharidoses (eg, Hurler syndrome, Scheie syndrome, Sanfilipposyndrome); Bardet-Biedl syndrome (Polydactyly, truncal obesity, kidneydysfunction, short stature, and pigmentary retinopathy); and neuronalceroid lipofuscinosis (Dementia, seizures, and pigmentary retinopathy;infantile form is known as Jansky-Bielschowsky disease, juvenile form isVogt-Spielmeyer-Batten disease, and adult form is Kufs syndrome).Retinitis pigmentosa is most commonly associated with mutations in theRHO, RP2, RPGR, RPGRIP1, PDE6A, PDE6B, MERTK, PRPH2, CNGB1, USH2A,ABCA4, BBS genes.

Diabetic retinopathy. Diabetic retinopathy (DR) is damage to the retinacaused by complications of diabetes, which can eventually lead toblindness. Without wishing to be bound by theory, it is believed thathyperglycemia-induced intramural pericyte death and thickening of thebasement membrane lead to incompetence of the vascular walls. Thesedamages change the formation of the blood-retinal barrier and also makethe retinal blood vessels become more permeable.

There are two stages of diabetic retinopathy: non-proliferative diabeticretinopathy (NPDR), and proliferative diabetic retinopathy (PDR).Nonproliferative diabetic retinopathy is the first stage of diabeticretinopathy, and is diagnosed by fundoscopic exam and coexistentdiabetes. In cases of reduced vision, fluorescein angiography may bedone to visualize the vessles in the back of the eye to and any retinalischemia that may be present. All people with diabetes are at risk fordeveloping NPDR, and as such, would be candidates for prophylactictreatment with the subject vectors. Proliferative diabetic retinopathyis the second stage of diabetic retinopathy, characterized byneovascularization of the retina, vitreous hemorrhage, and blurredvision. In some instances, fibrovascular proliferation causes tractionalretinal detachment. In some instances, the vessels can also grow intothe angle of the anterior chamber of the eye and cause neovascularglaucoma. Individuals with NPDR are at increased risk for developingPDR, and as such, would be candidates for prophylactic treatment withthe subject vectors.

Diabetic macular edema. Diabetic macular edema (DME) is an advanced,vision-limiting complication of diabetic retinopathy that affects nearly30% of patients who have had diabetes for at least 20 years, and isresponsible for much of the vision loss due to DR. It results fromretinal microvascular changes that compromise the blood-retinal barrier,causing leakage of plasma constituents into the surrounding retina and,consequently, retinal edema. Without wishing to be bound by theory, itis believed that hyperglycemia, sustained alterations in cell signalingpathways, and chronic microvascular inflammation with leukocyte-mediatedinjury leads to chronic retinal microvascular damage, which triggers anincrease in intraocular levels of VEGF, which in turn increases thepermeability of the vasculature.

Patients at risk for developing DME include those who have had diabetesfor an extended amount of time and who experience one or more of severehypertension (high blood pressure), fluid retention, hypoalbuminemia, orhyperlipidemia. Common symptoms of DME are blurry vision, floaters,double vision, and eventually blindness if the condition is allowed toprogress untreated. DME is diagnosed by funduscopic examination asretinal thickening within 2 disc diameters of the center of the macula.Other methods that may be employed include Optical coherence tomography(OCT) to detect retinal swelling, cystoid edema, and serous retinaldetachment; fluorescein angiography, which distinguishes and localizesareas of focal versus diffuse leakage, thereby guiding the placement oflaser photocoagulation if laser photocoagulation is to be used to treatthe edema; and color stereo fundus photographs, which can be used toevaluate long-term changes in the retina. Visual acuity may also bemeasured, especially to follow the progression of macular edema andobserve its treatment following administration of the subjectpharmaceutical compositions.

Retinal vein occlusions. A retinal vein occlusion (RVO) is a blockage ofthe portion of the circulation that drains the retina of blood. Theblockage can cause back-up pressure in the capillaries, which can leadto hemorrhages and also to leakage of fluid and other constituents ofblood.

Glaucoma. Glaucoma is a term describing a group of ocular (eye)disorders that result in optic nerve damage, often associated withincreased fluid pressure in the eye (intraocular pressure)(IOP). Thedisorders can be roughly divided into two main categories, “open-angle”and “closed-angle” (or “angle closure”) glaucoma. Open-angle glaucomaaccounts for 90% of glaucoma cases in the United States. It is painlessand does not have acute attacks. The only signs are graduallyprogressive visual field loss, and optic nerve changes (increasedcup-to-disc ratio on fundoscopic examination). Closed-angle glaucomaaccounts for less than 10% of glaucoma cases in the United States, butas many as half of glaucoma cases in other nations (particularly Asiancountries). About 10% of patients with closed angles present with acuteangle closure crises characterized by sudden ocular pain, seeing halosaround lights, red eye, very high intraocular pressure (>30 mmHg),nausea and vomiting, suddenly decreased vision, and a fixed, mid-dilatedpupil. It is also associated with an oval pupil in some cases.Modulating the activity of proteins encoded by DLK, NMDA, INOS, CASP-3,Bc1-2, or Bc1-xl may treat the condition.

Sorsby's fundus dystrophy. Sorsby's fundus dystrophy is an autosomaldominant, retinal disease associated with mutations in the TIMP3 gene.Clinically, early, mid-peripheral, drusen and colour vision deficits arefound. Some patients complain of night blindness. Most commonly, thepresenting symptom is sudden acuity loss, manifest in the third tofourth decades of life, due to untreatable submacularneovascularisation. Histologically, there is accumulation of a confluentlipid containing material 30 μm thick at the level of Bruch's membrane.

Vitelliform macular dystrophy. Vitelliform macular dystrophy is agenetic eye disorder that can cause progressive vision loss. Vitelliformmacular dystrophy is associated with the buildup of fatty yellow pigment(lipofuscin) in cells underlying the macula. Over time, the abnormalaccumulation of this substance can damage cells that are critical forclear central vision. As a result, people with this disorder often losetheir central vision, and their eyesight may become blurry or distorted.Vitelliform macular dystrophy typically does not affect side(peripheral) vision or the ability to see at night.

Researchers have described two forms of vitelliform macular dystrophywith similar features. The early-onset form (known as Best disease)usually appears in childhood; the onset of symptoms and the severity ofvision loss vary widely. It is associated with mutations in theVMD2/BEST1 gene. The adult-onset form (Adult vitelliform maculardystrophy) begins later, usually in mid-adulthood, and tends to causevision loss that worsens slowly over time. It has been associated withmutations in the PRPH2 gene. The two forms of vitelliform maculardystrophy each have characteristic changes in the macula that can bedetected during an eye examination.

Rod-cone dystrophy. Rod-cone dystrophies are a family of progressivediseases in which rod dysfunction, which leads to night blindness andloss of peripheral visual field expanses, is either the prevailingproblem or occurring at least as severely as cone dysfunction. Ascallop-bordered lacunar atrophy may be seen in the midperiphery of theretina. The macula is only mildly involved by clinical examinationalthough central retinal thinning is seen in all cases. Dyschromatopsiais mild early and usually becomes more severe. The visual fields aremoderately to severely constricted although in younger individuals atypical ring scotoma is present. The peripheral retina contains ‘whitedots’ and often resembles the retinal changes seen in retinitis punctatealbescens. Retinitis pigmentosa is the main group of diseases includedunder this definition and, as a whole, is estimated to affectapproximately one in every 3,500 people. Depending on the classificationcriteria used, about 60-80% of all retinitis pigmentosa patients have aclear-cut rod-cone dystrophy pattern of retinal disease and once othersyndromic forms are taken into account, about 50-60% of all retinitispigmentosas fall in the rod-cone dystrophy nonsyndromic category.

Leber's congenital amaurosis. Leber's congenital amaurosis (LCA) is asevere dystrophy of the retina that typically becomes evident in thefirst year of life. Visual function is usually poor and oftenaccompanied by nystagmus, sluggish or near-absent pupillary responses,photophobia, high hyperopia, and keratoconus. Visual acuity is rarelybetter than 20/400. A characteristic finding is Franceschetti'soculo-digital sign, comprising eye poking, pressing, and rubbing. Theappearance of the fundus is extremely variable. While the retina mayinitially appear normal, a pigmentary retinopathy reminiscent ofretinitis pigmentosa is frequently observed later in childhood. Theelectroretinogram (ERG) is characteristically “nondetectable” orseverely subnormal. Mutations in 17 genes are known to cause LCA: GUCY2D(locus name: LCA1), RPE65 (LCA2), SPATA7 (LCA3), AIPL1 (LCA4), LCAS(LCAS), RPGRIP1 (LCA6), CRX (LCAT), CRB1 (LCAS), NMNAT1 (LCAS), CEP290(LCA10), IMPDH1 (LCA 11), RD3 (LCAl2), RDH12 (LCA13), LRAT (LCA14),TULP1 (LCA15), KCNJ13 (LCA16), and IQCB1. Together, mutations in thesegenes are estimated to account for over half of all LCA diagnoses. Atleast one other disease locus for LCA has been reported, but the gene isnot known.

X-linked retinoschisis. X-linked retinoschisis (XLRS) is characterizedby symmetric bilateral macular involvement with onset in the firstdecade of life, in some cases as early as age three months. Fundusexamination shows areas of schisis (splitting of the nerve fiber layerof the retina) in the macula, sometimes giving the impression of a spokewheel pattern. Schisis of the peripheral retina, predominantlyinferotemporally, occurs in approximately 50% of individuals. Affectedmales typically have vision of 20/60 to 20/120. Visual acuity oftendeteriorates during the first and second decades of life but thenremains relatively stable until the fifth or sixth decade. The diagnosisof X-linked juvenile retinoschisis is based on fundus findings, resultsof electrophysiologic testing, and molecular genetic testing. RS 1 isthe only gene known to be associated with X-linked juvenileretinoschisis.

An individual affected by a cone cell disorder or at risk for developinga cone cell disorder can be readily identified using techniques todetect the symptoms of the disorder as known in the art, including,without limitation, fundus photography; Optical coherence tomography(OCT); adaptive optics (AO); electroretinography, e.g. ERG, color ERG(cERG); color vision tests such as pseudoisochromatic plates (Ishiharaplates, Hardy-Rand-Ritter polychromatic plates), the Farnsworth-Munsell100 hue test, the Farnsworth's panel D-15, the City university test,Kollner's rule, and the like; and visual acuity tests such as the ETDRSletters test, Snellen visual acuity test, visual field test, contrastsensitivity test, and the like; as will be known by the ordinarilyskilled artisan. Additionally or alternatively, the individual affectedby a cone cell disorder or at risk for developing a cone cell disordercan be readily identified using techniques to detect gene mutations thatare associated with the cone cell disorder as known in the art,including, without limitation, PCR, DNA sequence analysis, restrictiondigestion, Southern blot hybridization, mass spectrometry, etc. In someembodiments, the method comprises the step of identifying the individualin need of a cone cell therapy. In such instances, any convenient methodfor determining if the individual has the symptom(s) of a cone celldisorder or is at risk for developing a cone cell disorder, for exampleby detecting the symptoms described herein or known in the art, bydetecting a mutation in a gene as herein or as known in the art, etc.may be utilized to identify the individual in need of a cone celltherapy.

In practicing the subject methods, the subject composition is typicallydelivered to the retina of the subject in an amount that is effective toresult in the expression of the transgene in the cone cells. In someembodiments, the method comprises the step of detecting the expressionof the transgene in the cone cells.

There are a number of ways to detect the expression of a transgene, anyof which may be used in the subject embodiments. For example, expressionmay be detected directly, i.e. by measuring the amount of gene product,for example, at the RNA level, e.g. by RT-PCR, Northern blot, RNAseprotection; or at the protein level, e.g. by Western blot, ELISA,immunohistochemistry, and the like. As another example, expression maybe detected indirectly, i.e. by detecting the impact of the gene producton the viability or function of the cone photoreceptor in the subject.For example, if the gene product encoded by the transgene improves theviability of the cone cell, the expression of the transgene may bedetected by detecting an improvement in viability of the cone cell, e.g.by fundus photography, Optical coherence tomography (OCT), AdaptiveOptics (AO), and the like. If the gene product encoded by the transgenealters the activity of the cone cell, the expression of the transgenemay be detected by detecting a change in the activity of the cone cell,e.g. by electroretinogram (ERG) and color ERG (cERG); functionaladaptive optics; color vision tests such as pseudoisochromatic plates(Ishihara plates, Hardy-Rand-Ritter polychromatic plates), theFarnsworth-Munsell 100 hue test, the Farnsworth's panel D-15, the Cityuniversity test, Kollner's rule, and the like; and visual acuity testssuch as the ETDRS letters test, Snellen visual acuity test, visual fieldtest, contrast sensitivity test, and the like, as a way of detecting thepresence of the delivered polynucleotide. In some instances, both animprovement in viability and a modification in cone cell function may bedetected.

In some embodiments, the subject method results in a therapeuticbenefit, e.g. preventing the development of a disorder, halting theprogression of a disorder, reversing the progression of a disorder, etc.In some embodiments, the subject method comprises the step of detectingthat a therapeutic benefit has been achieved. The ordinarily skilledartisan will appreciate that such measures of therapeutic efficacy willbe applicable to the particular disease being modified, and willrecognize the appropriate detection methods to use to measuretherapeutic efficacy. For example, therapeutic efficacy in treatingmacular degeneration may be observed as a reduction in the rate ofmacular degeneration or a cessation of the progression of maculardegeneration, effects which may be observed by, e.g., fundusphotography, OCT, or AO, by comparing test results after administrationof the subject composition to test results before administration of thesubject composition. As another example, therapeutic efficacy intreating a progressive cone dysfunction may be observed as a reductionin the rate of progression of cone dysfunction, as a cessation in theprogression of cone dysfunction, or as an improvement in cone function,effects which may be observed by, e.g., ERG and/or cERG; color visiontests; functional adaptive optics; and/or visual acuity tests, forexample, by comparing test results after administration of the subjectcomposition to test results before administration of the subjectcomposition and detecting a change in cone viability and/or function. Asa third example, therapeutic efficacy in treating a color visiondeficiency may be observed as an alteration in the individual'sperception of color, e.g. in the perception of red wavelengths, in theperception of green wavelengths, in the perception of blue wavelengths,effects which may be observed by, e.g., cERG and color vision tests, forexample, by comparing test results after administration of the subjectcomposition to test results before administration of the subjectcomposition and detecting a change in cone viability and/or function.

Expression of the transgene using the subject transgene is expected tobe robust. Accordingly, in some instances, the expression of thetransgene, e.g. as detected by measuring levels of gene product, bymeasuring therapeutic efficacy, et.c, may be observed two months or lessafter administration, e.g. 4, 3 or 2 weeks or less after administration,for example, 1 week after administration of the subject composition.Expression of the transgene is also expected to persist over time.Accordingly, in some instances, the expression of the transgene, e.g. asdetected by measuring levels of gene product, by measuring therapeuticefficacy, etc., may be observed 2 months or more after administration ofthe subject composition, e.g., 4, 6, 8, or 10 months or more, in someinstances 1 year or more, for example 2, 3, 4, or 5 years, in certaininstances, more than 5 years.

In certain embodiments, the method comprises the step of detectingexpression of the transgene in the cone cells, wherein expression isenhanced relative to expression from a polynucleotide cassette notcomprising the one or more improved elements of the present disclosure,i.e. a reference control, e.g. the pR2.1 promoter or variants thereof(e.g. pR1.7, pR1.5, pR1.1, etc.) as disclosed in, e.g., US ApplicationNo. 2013/0317091, or the synthetic IRBP/GNAT2 promoter as disclosed inUS Application No. 2014/0275231; the full disclosures of which areincorporated herein by reference. Typically, expression will be enhanced2-fold or more relative to the expression from a reference, i.e. acontrol polynucleotide cassette, e.g. as known in the art, for example3-fold, 4-fold, or 5-fold or more, in some instances 10-fold, 20-fold or50-fold or more, e.g. 100-fold, as evidenced by, e.g. earlier detection,higher levels of gene product, a stronger functional impact on thecells, etc.

Typically, if the subject composition is an rAAV comprising the subjecta polynucleotide cassette of the present disclosure, an effective amountto achieve a change in will be about 1×10⁸ vector genomes or more, insome cases 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ vector genomes ormore, in certain instances, 1×10¹⁴ vector genomes or more, and usuallyno more than 1×10¹⁵ vector genomes. In some cases, the amount of vectorgenomes that is delivered is at most about 1×10¹⁵ vector genomes, e.g.1×10¹⁴ vector genomes or less, for example 1×10¹³, 1×10¹², 1×10¹¹,1×10¹⁰, or 1×10⁹ vector genomes or less, in certain instances 1×10⁸vector genomes, and typically no less than 1×10⁸ vector genomes. In somecases, the amount of vector genomes that is delivered is 1×10¹⁰ to1×10¹¹ vector genomes. In some cases, the amount of vector genomes thatis delivered is 1×10¹⁰ to 3×10¹² vector genomes. In some cases, theamount of vector genomes that is delivered is 1×10⁹ to 3×10¹³ vectorgenomes. In some cases, the amount of vector genomes that is deliveredis 1×10⁸ to 3×10¹⁴ vector genomes.

In some cases, the amount of pharmaceutical composition to beadministered may be measured using multiplicity of infection (MOI). Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷ and 1×10¹⁸ MOI.

In some aspects, the amount of pharmaceutical composition comprisesabout 1×10⁸ to about 1×10¹⁵ particles of recombinant viruses, about1×10⁹ to about 1×10¹⁴ particles of recombinant viruses, about 1×10¹⁰ toabout 1×10¹³ particles of recombinant viruses, or about 1×10¹¹ to about3×10¹² particles of recombinant viruses.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the subject composition or itsby-products, and thus based on the disposition of the composition withinthe subject. This includes consideration of the route of administrationas well as dosage amount, which can be adjusted for subretinal (applieddirectly to where action is desired for mainly a local effect),intravitreal (applied to the vitreaous for a pan-retinal effect), orparenteral (applied by systemic routes, e.g. intravenous, intramuscular,etc.) applications. Effective amounts of dose and/or dose regimen canreadily be determined empirically from preclinical assays, from safetyand escalation and dose range trials, individual clinician-patientrelationships, as well as in vitro and in vivo assays such as thosedescribed herein and illustrated in the Experimental section, below.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Background

New therapies are needed for the treatment of many cone photoreceptorassociated disorders, including macular dystrophies such as cone-roddystrophy, cone dystrophy, Stargardt macular dystrophy, andachromatopsia; color vision disorders such as protan, deutan, and tritandefects; and vision disorders of the central macula such as age-relatedmacular degeneration, macular telangiectasia, retinitis pigmentosa,diabetic retinopathy, retinal vein occlusions, glaucoma, Sorsby's fundusdystrophy, adult vitelliform macular dystrophy, Best's disease, andX-linked retinoschisis. As these vision disorders are associated with aloss of function and/or viability of the cone photoreceptors, it ishypothesized that these disorders may be treatable by delivering atherapeutic gene to cone photoreceptors to rescue cone viability andfunction.

To that end, the polynucleotide cassette “pMNTC” was designed in whichenhancer, promoter, 5′UTR, intron, Kozak, and polyadenylation sequenceswere designed for cone-specific expression (FIG. 10 a). The cassetteincluded an LCR enhancer sequence from the L- and M-opsin genomic locusand a truncated promoter sequence from the M-Opsin gene, comprisingabout 140 nucleotides upstream of the transcriptional start site. Inaddition, the cassette included a 5′ untranslated region (5′ UTR) basedon the M-opsin 5′UTR but modified to have minimal secondary structure(see FIG. 3) and to include additional sequence at its 3′ end into whichan intron was inserted. The intronic sequence used was a pSI chimericintron having the 5′-donor site from the first intron of the humanβ-globin gene and the branch and 3′-acceptor site from the intron thatlies between the leader and the body of an immunoglobulin gene heavychain variable region (Bothwell, A. L. et al. (1981) Heavy chainvariable region contribution to the NPb family of antibodies: Somaticmutation evident in a gamma 2a variable region. Cell 24, 625-37). Thesequences of the donor and acceptor sites, along with the branchpointsite, were changed to match the consensus sequences for splicing(Senapathy, P., Shapiro, M. B. and Harris, N. L. (1990) Meth. Enzymol.183, 252-78). Also included in the pMNTC polynucleotide cassette was astrong Kozak sequence and an SV40 polyadenylation sequence.

Experiments were also performed to identify the best AAV with which todeliver transgenes to cone cells. Successful delivery of polynucleotidesto cells of the retina for the purposes of gene therapy has beenachieved using viral vectors such as AAV and lentivirus. However, theseviruses must be injected subretinally to reach the cells of thenon-human primate (NHP) retina, a procedure that carries with it therisk of retinal damage. A less disruptive approach is administration byintravitreal injection. However, efficient transduction of conephotoreceptors following intravitreal delivery of AAV or lentivirus hasnever been demonstrated: while reports exist of AAVs with the ability totransduce retinal cone cells with high efficiency (Merigan et al. IOVS2008,49 E-abstract 4514), later reports have questioned the efficacy ofthese vectors (Yin et al. IOVS 2011, 52(5):2775-2783).

Results

Directed evolution of AAV2 has led to the identification of the viralvariant “7m8” that is able to transduce photoreceptors better than wildtype AAV2 (Dalkara et al. Sci Transl Med 2013). However, the retinacontains two types of photoreceptors—rods and cones—and no reports existdemonstrated whether AAV2-7m8 can transduce cone photoreceptors, per se,and more particularly, cone photoreceptors in the highly cone-enrichedarea of the fovea. To test this possibility, we delivered AAV2-7m8carrying an expression cassette of the ubiquitous promoter CMV operablylinked to GFP to the retina of African Green monkey by intravitrealinjection. Intravitreally delivered AAV2-7m8.CMV.GFP appeared totransduce retinal cells in the fovea centralis (the 0.35 mm diameterrod-free region of retina at the center of the foveal pit) and parafovea(the lip of the depression) of primates more efficiently thanintravitreally-delivered AAV2 or other AAV variants previously shown inthe art to transduce retinal cells. Neither AAV2-7m8 nor the other AAVstested tested appeared to be able to transduce the cones of the primatefovea, the 1 5 mm-diameter cone-enriched region of retina that surroundsthe foveola and forms the slopes of the pit (FIG. 5).

We next packaged a genome comprising pMNTC operably linked to GFP withinthe AAV2-7m8 capsid, and assessed the ability of this vector compositionto express the GFP transgene in cone cells in vivo when injectedintravitreally. Expression was evaluated in a number of species withvarying numbers of retinal cones cells among total photoreceptors,including mouse (3% cones), rat (1% cones), gerbil (13% cones), andnonhuman primate (5% cones). Contrary to our results in FIG. 5, stronggene expression could be detected throughout the nonhuman primate fovea(FIG. 6). These data indicate that intravitreally delivered AAV2-7m8can, in fact, transduce retinal cones, and that pMNTC acts as a robustexpression cassette in cone cells. Robust reporter gene expression wasalso seen in the intravireally injected retina of the rat (data notshown) and gerbil (FIG. 8A), with expression levels and anatomiclocation correlating with cone abundance and location in all species.

To determine the cell-specificity of pMNTC-directed expression, wholemounts of transduced mouse retina were analyzed by immunohistochemistryusing an antibody that is specific for cone L and M opsins. Theexpression of L/M opsin, which labels the outer segments of conephotoreceptors only, was observed in virtually all of the cones of themouse retina that expressed GFP from the AAV2-7m8.MNTC.GFP vector (FIG.7), indicating that MNTC-directed expression of transgenes is highlycone-specific. Moreover 80% or more of the cone outer segments that werelabelled by the L/M opsin-specific antibody also expressed the GFPtransgene, indicating that AAV2-7m8 transduces cones highly efficiently(FIG. 7).

We next compared the ability of pMNTC to promote expression in conecells to that of pR2.1. pR2.1 comprises the human L/M opsin enhancer(“LCR”) and the promoter region from the human L-Opsin gene. Inaddition, pR2.1 comprises the L-Opsin 5′UTR fused to additional 5′UTRsequence at its 3′ end, into which modified SV40 late 16s intronicsequence has been inserted. This is followed by the L-Opsin Kozaksequence, which is then typically linked in-frame to a transgene. At theend of the cassette is an SV40 polyA tail.

Viral preparations of AAV2-7m8.MNTC.GFP and AAV2-7m8.pR2.1.GFP weredelivered intravitreally to the retinas of gerbils and nonhuman primatesin vivo, and the retinas imaged in vivo 2 weeks, 4 weeks, 8 weeks, and12 weeks later by fundus autofluorescence and OCT. GFP reporterexpression was detected sooner, more strongly, and in more cones ingerbil retina transduced with rAAV carrying the pMNTC.GFP expressioncassette than in gerbil retinas carrying the pR2.1.GFP expressioncassette (FIG. 8B). Likewise, GFP reporter expression was detectedsooner and in more cones in nonhuman primate retinas transduced withrAAV carrying the pMNTC.GFP expression cassette as compared to NHPretinas transduced with the pR2.1 expression cassette (FIG. 9, n=4eyes). In both gerbils and NHP, GFP was consistently observed to bestronger from pMNTC than from pR2.1 throughout the duration of thestudy.

To determine the contribution of each of the elements in the pMNTCexpression cassette to the overall improvement in expression, a seriesof expression constructs were cloned in which each of the elements inpMNTC was substituted one-by-one with the corresponding element from thepR2.1 expression cassette. These constructs were then packaged intoAAV2-7m8 and delivered by intravitreal injection to the gerbil retina.Gerbil retinas were assessed 4 and 8 weeks later in vivo by in vivobioluminescence (IVIS imaging system, PerkinElmer), which provides aquantitative readout of reporter expression across the entire eye.

As expected, expression of the luciferase reporter under the control ofpMNTC was higher than expression of the luciferase reporter under thecontrol of pR2.1. Replacement of the pMNTC promoter sequence with thepR2.1 promoter sequence having the most sequence homology to it (SEQ IDNO:83) reduced expression (construct pMNTC pR2.1 L3 ‘P), as did theinclusion of pR2.1 promoter sequence that lies more distal to the 5′UTRof pR2.1 (SEQ ID NO:82) (construct pMNTCpR2.1-L5′P). Expression was alsoreduced by the introduction into the pMNTC 5′UTR of two false startsequences (“AUG1” and “AUG2”) that were observed in the pR2.1 5′UTR(construct pMNTC_(—)2.1-AUG1/2). Interestingly, expression was notreduced when the pMNTC 5′UTR was replaced with a modified pR2.1 5′UTRsequence in which these false starts had been removed (SEQ ID NO:87,nucleotide 17 changed to C, nt 61and 62 changed to CA)(pMNTC_pR2.1-5′UTR), suggesting that the pR2.1 5′UTR would promotestrong expression in cone cells but for the false AUGs in the pR2.15′UTR element. Also interestingly, the pR2.1 intron (SEQ ID NO:59)appeared to provide more robust expression than the pSI chimeric intronof pMNTC, suggesting that inclusion of the pR2.1 intron in thepolynucleotide cassettes of the present disclosure may be used tofurther improve expression in cone cells. Lastly, removal of the L/Menhancer (found in both pR2.1 and pMNTC) reduced expression as well.While the polyA tailed seemed at first to also have a significant impacton expression, re-sequencing of the pMNTC construct comprising thispR2.1 element revealed that the polyA tail was not operably linked tothe transgene, thereby explaining why only background levels ofexpression were observed from this construct. Thus, the L/M opsin LCR,the inclusion of the M opsin core promoter rather than the L opsinpromoter, and the exclusion of false starts in the 5′UTR all contributeto the enhancement in gene expression achieved using the pMNTC promoter.

In conclusion, we have identified an AAV variant, the AAV variantcomprising a 7m8 peptide in the GH loop, which may be used for theintravitreal delivery of polynucleotides to retinal cones. Likewise, wehave identified a number of polynucleotide cassette elements that may beused to promote strong expression in cone photoreceptors. Together,these discoveries represent improvements that may facilitate thedevelopment of therapeutic agents for cone-associated disorders.

Materials and Methods

Transgene expression in vitro in WERI-RB-1 cells. WERI-Rb-1retinoblastoma cells expressing cone photoreceptor pigments cells aretransfected with a polynucleotide cassette of the present disclosureaccording to the method described by Shaaban and Deeb, 1998; IOVS39(6)885-896. The polynucleotide cassettes are transfected as plasmidDNA using well established techniques of molecular biology, such ascloning (Maniatis et al.) or via de novo DNA synthesis. All regulatoryelements are placed in the cassette and used to drive the enhanced GFPprotein. Plasmid DNA is then introduced into cells using establishedtechniques for non-viral transfection, for example using a lipid-basedtransfection reagent (Altogen Biosystems, NV) or Lipofectamine LTX (LifeTechnologies). Cells are then cultured for 72 hours and eGFP expressionis measured using flow cytometry and fluorescence microscopy. Transgeneexpression in cells transfected with the polynucleotide cassette of thepresent invention (i.e., constructs designed for cone photoreceptorexpression) is compared to the un-optimized counterparts (i.e., thosebased on pR2.1) and is found to be stronger from cassettes carryingimproved elements

In vitro expression is also evaluated using other mammalian cell linesthat express cone opsins, such as 661W cells (Tan et al., IOVS 2004;45(3) 764-768).

Similarly, in vitro expression is evaluated using non-photoreceptor celllines that have been engineered to express cone photoreceptor-specificproteins. Such a system has been described with HEK293 cells that havebeen genetically engineered to express CRX/Spl (Khani et al., IOVS 2007;48: 3954). Marker genes are also used (eGFP, dsRed, mCherry, luciferase)as well as physiologic genes (opsin, ACHR genes). Physiologic genes aretested by examining mRNA levels (e.g., by RT-PCR) or protein levels(e.g., by ELISA or Western blot).

Animal care. All experiments conformed to the principles regarding thecare and use of animals adopted by the American Physiological Societyand the Society for Neuroscience, and were approved by the InstitutionalAnimal Care and Use Committee (IACUC).

Small animal studies. The expression of the gene product encoded by thecoding sequence of the expression cassettes are evaluated in vivo inmice, rats, and gerbils. This is accomplished by intravitreal injectionin vivo of an rAAV preparation comprising the expression cassette (Li etal., 2008; Mol Vis 48: 332-338). Note that electroporation of plasmidDNA may be performed instead (Matsuda/Cepko).

Mouse studies. Mice used in this study were C57BL/6. Animals wereanesthetized with ketamine/xylazine (110 mg/kg intraperitoneal). Abeveled 34 gauge disposable needle loaded with test article was insertedinto the vitreous of the eye, and 5.04×10¹⁰ vector genomes of rAAV in avolume of 1.5 μl was injected into the vitreous.

Gerbil and rat studies. Mongolian gerbils (Meriones unguiculatus) andbrown Norway rats were used in this study. Pupils were dilated with 10%phenylephrine and 0.5% tropicamide. Animals were anesthetized with anintraperitoneal or intramuscular injection of 0.1-0.2 mL of aketamine/xylazine solution (70 mg/mL ketamine and 10 mg/mL xylazine forrats; 25 mg/mL ketamine and 0.3 mg/mL xylazine for gerbils). A beveled34 gauge disposable needle loaded with test article in a 100 μL Hamiltonsyringe was inserted into the vitreous of the eye through the sclera atan optimized superior-temporal point about 1 mm from Limbus.1×10¹⁰-2×10¹⁰ vector genomes of test article (2×10¹⁰ vg of rAAV.GFP, or1.15×10¹⁰ vg of rAAV.luciferase) in a 5 uL volume was injected slowlywith a micro-injection pump into the vitreous, after which the needletip was held in the injected eye at the injected position for 10 secondsso as to ensure adequate test article dispensing. The needle was thenwithdrawn.

Non-human primate (NHP) studies. The polynucleotide cassettes andexpression vectors are also tested in large animals. This is done byusing AAV, for example using the techniques of Mancuso et al. Briefly,an AAV cassette is made, the AAV encapsidating the expression cassetteis manufactured, and the viral prep is injected intravitreally (up to170 uL in the vitreous) or subretinally (up to 3, 100 uL injections atdifferent locations; vitrectomy may be performed prior to injection) innonhuman primates. Expression is evaluated by reporter (GFP), color ERG,and/or behavioral testing using the Cambridge Color Test or on animalstrained to make a saccade (eye movement) when a target enters the fieldof view. The saccades are monitored using an eye tracker. Prior totreatment animals are trained to perform a color vision test or to makea saccade when it sees a colored target. An ERG is performed to estimatethe spectral sensitivity of the cones present. Data from the colorvision test performance and the ERG provide evidence that the animal isdichromatic (colorblind). For animals that receive a vector carrying theGFP gene, expression is monitored using fundus imaging with RetCam II orsimilar device under light that produces excitation of the GFP. Foranimals receiving a photopigment gene that differs in spectralsensitivity compared to the animal's endogenous pigments, expression ismonitored using the multifocal color ERG to measure spectral sensitivityat up to 106 different retinal locations, and by behavioral testing.

Baboons were sedated with 10-15 mg/kg ketamine following bysevofluorane. African Green monkeys were sedated with an intramuscularinjection of 5:1 ketamine:xylazine mix (0.2 ml/kg of 100 mg/ml ketamineand 20 mg/ml xylazine). Mydriasis was achieved with topical 10%phenylephrine. An eye speculum was placed in the eye to facilitateinjections. A drop of proparacaine hydrochloride 0.5% and then 5%betadine solution was applied, followed by a rinse with sterile saline.Baboons (FIG. 6) received 60 μl of a 3.4×10¹³ vg preparation of rAAV byintravitreal (ITV) injection to yield a final dose of 2.02×10¹² vg pereye. African Green monkeys received 50 uL of a 1×10¹³ preparation ofrAAV vector by ITV injection to yield a final dose of 5×10¹¹ vg per eye.ITV injections to the central vitreous were administered using a31-gauge 0.375 inch needle (Terumo) inserted inferotemporally at thelevel of the ora serrata ˜2 5 mm poster to the limbus under a surgicalmagnification to allow full visualization of extraocular and intraocularneedle placement. Central vitreous placement was confirmed by directobservation of the needle tip at the time of the injection. FollowingITV injections a topical triple antibiotic ointment was administered.

Slit-lamp biomicroscopy. The anterior segment of each monkey eye wasexamined by slit-lamp biomicroscopy during baseline screening and atweek 4 (day 28), week 8 (day 56) and week 12 (day 84) post-injection tomonitor inflammation. No abnormalities were observed.

Fundus examination and photography. Eye examination and fundusphotography of rat and gerbil retinas was performed using a PhoenixMicron IV fundus microscope. All animals received a baselinescreening/photographing to confirm ocular health, and then photographedat the designated timepoints to monitor the expression of the GFPtransgene. Any change to the optic nerves and retina or appearance ofgross lesions were recorded by a color fundus photography and expressionof GFP was visualized using fluorescence fundus imaging with afluorescein filter.

Retinal examination, fundus color and fluorescence photography, andautofluorescence OCT of NHP were performed by using a Topcon TRC-50EXretinal camera with Canon 6D digital imaging hardware and New VisionFundus Image Analysis System software and Spectralis OCT Plus. Allanimals received a baseline imaging. GFP expression was also documentedat week 2, 4, 8, and 12 post-intravitreal vector injection.

IVIS Imaging System. Expression of luciferase in the retina followingdelivery of rAAV.luciferase was quantified in vivo 2, 4 and 8 weekspost-intravitreal injection using an IVIS Imaging System. Gerbils wereinjected subcutaneously with 150 mg/kg luciferin (PerkinElmer) (15 mg/mlluciferin at a dose of 15 ml/kg). Approximately 22 minutes later,animals were sedated by inhalation of 4% isoflurane for 3-5 minutes.Immediately thereafter, animals were placed on the imaging platform inpairs, and the luminescence of the one eye of each animal quantifiedfollowed immediately by imaging of the contralateral eye. A naïve gerbilwas used as a negative standard, with background levels of luminescencetypically registering a luminescence of 1×10⁴ photons/second.Bioluminescence verification using a phantom mouse (XPM-2 Perkin Elmerphantom mouse for bioluminescence imaging) was performed prior toimaging to ensure calibration of the imaging system.

Immunohistochemistry. Mice were euthanized with a lethal dose of sodiumpentobarbital and tissues fixed via cardiac perfusion first with 0.13Mphosphate buffered saline (PBS) pH 7.2-7.4 containing 2 units of heparinper mL, followed by 4% paraformaldehyde (PFA) in PBS, followed by 4%paraformaldehyde plus 1% glutaraldehyde in PBS. Glutaraldehyde served tokeep the neural retina attached to the RPE so that the cone outersegments would remain intact. Each solution was warmed to ˜37° C. justprior to administration and ˜35-40mL of perfusate was delivered at eachstage. Once the perfusion was stopped, the mouse was wrapped in a moistpaper towel and left to further fix for 2-3 hours before enucleation anddissection.

Permanent ink was used to mark the orientation of the eye, the anteriorsegment was removed, and the eye-cup was fixed in 4% PFA overnight at 4°C. and then stored in PBS at 4° C. Retinal whole-mounts were made byflattening the dissected retina between tissues soaked in 4% PFA for twohours and then transferring them to a culture plate for 6 more hours offixation. Afterward, the PFA was replaced with PBS containing 0.03%sodium azide (Sigma).

Antibody labeling was carried out on a rotating table shaker. To blocknon-specific labeling, whole mounts were incubated overnight at 4° C.with a solution containing 5% donkey serum (Jackson ImmunoResearch, Cat#004-000-120), 1 mg/ml BSA (Jackson ImmunoResearch, Cat #001-000-161),and 0.03% Triton X-100 in PBS (pH 7.4). The primary antibody used inthis study was rabbit anti red-green (L/M) opsin diluted 1:200(Millipore, Cat #AB5405. Specimens were washed in PBS 3 times for 30minutes each, then incubated at 4° C. overnight with DAPI(4′,6-diamidino-2-phenylindole, dihydrochloride 1:10,000; Invitrogen,Cat #D-21490) plus secondary antibodies. The secondary antibody for theL/M-opsin antibody was Alexa Fluor 488 labeled donkey anti-rabbitIgG(H+L) diluted 1:200 in antibody dilution buffer (Invitrogen, Cat #A21206). The incubation with secondary antibody was followed by three 30minute PBS washes, 30 minutes of post-fixation with 4% paraformaldehyde,and three more 30 minute PBS washes. Finally, the retinal slices wereplaced on slides with 2% DABCO in glycerol and covered with cover slips.

Microscopy. Widefield images of mouse retina whole mounts were acquiredusing a Nikon Eclipse E1000 with a 20× (open-air) objective and cameraset with a 1.5× optical zoom. For each specimen, 50 optical sectionswere taken 0.5 μm apart and the M-opsin Z-stack was reconstructed inImageJ. The Z-stack was oriented so that the lengths of the outersegments were in plane, and the distance between where antibody stainingbegan and ended was measured as an estimate of the length of the outersegments. Further, a 3D projection of the Z-stack was generated and thenumber of cones with visible M-opsin in the outer segment could bequantified.

Confocal image slices were acquired using an Olympus FluoView™ FV1000.Sections were imaged using a 20× oil immersion lens (40 images taken 0.5μm apart) and the Z-stacks were reconstructed in ImageJ. Channelexposure levels were balanced within and across images using AdobePhotoshop. For the retinal whole mounts, images were taken using a 10×open-air lens and mosaics were constructed with Adobe Photoshop's nativemosaic construction software.

Experiments testing the tissue specificity of the polynucleotidecassettes. In this instance, a construct encoding GFP is injected viaone or more routes of administration, such as intravitreal, subretinal,or intravenously. The animal is then sacrificed and tissues are analyzedby qPCR—to detect DNA sequences indicating presence of the construct—andGFP expression—to detect areas where the construct is activelyexpressed. Whereas absence of DNA sequence indicates lack ofbiodistribution to a given tissue, the presence of DNA sequence togetherwith the lack of transgene expression (mRNA or protein level) indicatespresence of vector but lack of expression in that tissue. In this way,the level of specificity for cone photoreceptors can be established, andused to determine the utility of this invention in terms of restrictingexpression to target cone photoreceptor cells without expression innon-targeted tissues such as optic nerve, liver, spleen, or braintissue. Intravitreal AAV is known to biodistribute to the brain (Provostet al) so highly expressed, improved constructs for targeting conephotoreceptors would be useful to limit expression to target cells ofthe retina and limit potential adverse events associated with off-targettransgene expression.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

1. A polynucleotide cassette for enhanced expression of a transgene incone cells of a mammalian retina, comprising (a) a promoter region,wherein the promoter region is specific for retinal cone cells; (b) acoding sequence operatively linked to the promoter region; and (c) apolyadenylation site.
 2. The polynucleotide cassette of claim 1, whereinthe promoter region comprises a polynucleotide sequence having asequence identity of 85% or more to a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:53, SEQID NO:54, SEQ ID NO:55, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81. SEQ IDNO:82, and SEQ ID NO:83, or a functional fragment thereof.
 3. Thepolynucleotide cassette of claim 2, wherein the promoter region consistsessentially of a polynucleotide sequence having a sequence identity of85% or more to the full length of SEQ ID NO:55 or a functional fragmentthereof.
 4. The polynucleotide cassette of claim 1, comprising apolynucleotide sequence encoding an untranslated region 5′ of the codingsequence.
 5. The polynucleotide cassette of claim 4, wherein thepolynucleotide sequence encoding an untranslated region 5′ of the codingsequence comprises a sequence having a sequence identity of 85% or moreto a sequence selected from the group consisting of SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87, SEQ ID NO:88, and SEQ ID NO:89, or a fragment thereof.
 6. Thepolynucleotide cassette of claim 4, wherein the untranslated region 5′of the coding sequence does not comprise a polynucleotide ATG.
 7. Thepolynucleotide cassette of claim 4, wherein the polynucleotide sequenceencoding an untranslated region 5′ of the coding sequence consistsessentially of a sequence having a sequence identity of 85% or more tothe full length of SEQ ID NO:85 or SEQ ID NO:86, or a fragment thereof.8. The polynucleotide cassette of claim 1, further comprising an intron.9. The polynucleotide cassette of claim 8, wherein the intron comprisesa sequence having a sequence identity of 85% or more to a sequenceselected from the group consisting of SEQ ID NO:5, SEQ ID NO:59, and SEQID NO:60.
 10. The polynucleotide cassette of claim 9, wherein the intronis located within the polynucleotide sequence encoding an untranslatedregion 5′ of the coding sequence.
 11. The polynucleotide cassette ofclaim 1, further comprising a translation initiation sequence.
 12. Thepolynucleotide cassette of claim 11, wherein the translation initiationsequence comprises polynucleotide sequence consisting essentially of SEQID NO:72 or SEQ ID NO:73.
 13. The polynucleotide cassette of claim 1,further comprising an enhancer sequence having a sequence identity of85% or more to SEQ ID NO:52 or a functional fragment thereof.
 14. Thepolynucleotide cassette of claim 13, wherein the enhancer sequenceconsists essentially of a sequence having a sequence identity of 85% ormore to the full length of SEQ ID NO:51.
 15. The polynucleotide cassetteof claim 1, wherein expression of the coding sequence is greater thanexpression of the coding sequence when the promote region is replacedwith the promoter regions of SEQ ID NO:1 when introduced into amammalian cone cell.
 16. A recombinant adeno-associated virus (rAAV)comprising: a) an AAV capsid protein, and b) the polynucleotide cassetteclaim 1 flanked by AAV ITRs.
 17. A pharmaceutical composition comprisingthe rAAV of claim 16 and a pharmaceutical excipient.
 18. A method forexpressing a transgene in cone cells, comprising: contacting one or morecone cells with an effective amount of the recombinant adeno-associatedvirus of claim 16, wherein the transgene is expressed at detectablelevels in the one or more cone cells.
 19. The method according to claim18, comprising detecting the expression in the cone cells, whereinexpression is detected in 60% or more of the cone cells.
 20. The methodaccording to claim 19, wherein the transgene is expressed at detectablelevels specifically in cone cells.
 21. A method for the treatment orprophylaxis of a cone cell disorder in a mammal in need thereof,comprising administering to the eye of the mammal an effective amount ofthe pharmaceutical composition of claim 17, wherein the coding sequenceencodes a therapeutic gene product.
 22. The method of claim 21, whereinthe cone cell disorder is a macular dystrophy, a color vision disorder,or a vision disorder of the central macula.
 23. The method of claim 22,wherein the color vision disorder is selected from the group consistingof achromotopsia, blue cone monochromasy, a protan defect, a deutandefect, and a tritan defect.
 24. The method of claim 22, wherein themethod further comprises detecting a change in the disease symptoms,wherein the change comprises an increase in the ability of the mammal toperceive a color.
 25. The method of claim 22, wherein the maculardystrophy is selected from the group consisting of Stargardt's maculardystrophy, cone dystrophy, cone-rod dystrophy, Spinocerebellar ataxiatype 7, and Bardet-Biedl syndrome-1.
 26. The method of claim 22, whereinthe vision disorder of the central macula is selected from the groupconsisting of age-related macular degeneration, macular telangiectasia,retinitis pigmentosa, diabetic retinopathy, retinal vein occlusions,glaucoma, Sorsby's fundus dystrophy, adult vitelliform maculardystrophy, Best's disease, rod-cone dystrophy, Leber's congenitalamaurosis, and X-linked retinoschisis.
 27. The method of claim 22,wherein the method further comprises detecting a change in the diseasesymptoms, wherein the change comprises reducing the rate of visualacuity loss of the mammal. 28-57. (canceled)